Troponin and bnp based diagnosis of risk patients and cause of stroke

ABSTRACT

Method for determination of whether a subject suffers from intermittent atrial fibrillation based on the determination of the amount of a Troponin T in a sample in a patient suffering from atrial fibrillation using a binding assay. Kits and devices adapted to carry out the methods are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/251,803 filed Apr. 14, 2014, which is a continuation of InternationalApplication No. PCT/EP2012/070553, filed Oct. 17, 2012, which claims thebenefit of European Patent Application No. 11185421.2, filed Oct. 17,2011, the disclosures of which are hereby incorporated by reference intheir entirety.

BACKGROUND OF THE DISCLOSURE

Stroke ranks after ischemic heart disease second as a cause of lostdisability—adjusted life—years in high income countries and as a causeof death worldwide. If presented early adverse consequences of strokecan be ameliorated using thrombolysis, in case of late presentationsecondary prevention (to prevent subsequent stroke) using aspirin andanticoagulation appears the only appropriate method to avoid diseaseprogression (van der Worp B and van Gijn J., NEJM 2007: 357: 572-578).

In order to prevent or treat stroke the identification of the underlyingcause of stroke is of importance. This has been addressed by the TOASTcriteria (Adams H. P. et al Stroke 1993: 24: 35-41). TOAST criteriadissect causes of stroke into atherothrombotic (atherosclerosis of largevessels), cardioembolic, lacunar (involving small vessels) andundetermined (Adams H. P. et al). In order to assess these criteriacarotid and transcranial ultrasound are required as well asechocardiography and an electrocardiogram (Rodriguez-Yanez et al,Disease Markers 2009: 26: 189-195).

Cardiac troponins T and I are the preferred biomarkers for the diagnosisof acute myocardial infarction (Anderson J L, ACC/AHA 2007 guidelinesfor the management of patients with unstable angina/non-ST-Elevationmyocardial infarction. J Am Coll Cardiol. 2007; 50(7):e1-e157). It hasbeen recognized that elevated troponin levels may be detected in severalnon-acute chronic disease states, including coronary artery disease,heart failure, and chronic kidney disease (see e.g. Omland et al., NEngl J Med. 2009; 361(26):2538-2547). Troponins T and I are also beenshown to be detectable in individuals from the general population (seee.g. Wallace et al., Prevalence and determinants of troponin T elevationin the general population. Circulation. 2006; 113(16):1958-1965).

SUMMARY OF THE DISCLOSURE

The present disclosure relates to a method for early differentiatingwhether a subject suffers from cardioembolic stroke or fromnon-cardioembolic ischemic stroke. The method is based on thedetermination of the amount of a cardiac Troponin in a sample from asubject suffering from ischemic stroke obtained not more than 24 hoursafter the onset of symptoms of ischemic stroke. Moreover, the presentdisclosure relates to a method for diagnosing atrial fibrillation in asubject. Further envisaged by the present disclosure are kits anddevices adapted to carry out the method of the present disclosure. Thepresent disclosure also relates to a system for early differentiatingwhether a subject suffers from cardioembolic stroke or fromnon-cardioembolic ischemic stroke and for diagnosing atrialfibrillation. Further, the present disclosure relates to reagents andkits used in performing the methods disclosed herein.

In the context of the present disclosure, it has been surprisingly shownthat elevated cardiac Troponin levels in patients with cardioembolicstroke are detectable already at the onset of cardioembolic stroke.Thus, the increase of the level of cardiac Troponins is not caused bythe stroke event. Rather, the studies of the present disclosure suggestthat the levels of cardiac troponins are already increased before theonset of stroke symptoms. Therefore, the determination of cardiacTroponins allows for an early differentiation between cardioembolicischemic stroke and non-cardioembolic ischemic stroke in the subject.This is advantageous since the early assessment of the cause of strokeis crucial in order to sufficiently treat a subject suffering fromstroke, in particular a subject suffering from cardioembolic stroke.Moreover, strokes due to cardioembolism are in general severe and proneto early recurrence

DETAILED DESCRIPTION OF THE EMBODIMENTS OF THE DISCLOSURE

The embodiments disclosed herein are not intended to be exhaustive orlimit the disclosure to the precise form disclosed in the followingdetailed description. Rather, the embodiments are chosen and describedso that others skilled in the art may utilize their teachings.

Conventional diagnostic techniques usually do not allow for an early,reliable assessment of the cause of stroke. Accordingly, a personalizedtreatment regimen cannot be determined with sufficient accuracy. As aconsequence thereof, many patients will receive a treatment regimenwhich is insufficient or which may have adverse side effects. Therefore,means and methods are required for reliably differentiating between thecauses of stroke.

Accordingly, the present disclosure method for early differentiatingwhether a subject suffers from cardioembolic stroke or fromnon-cardioembolic ischemic stroke, comprising

-   -   a) determining the amount of a cardiac Troponin in a sample from        a subject suffering from ischemic stroke,    -   wherein the sample has been obtained immediately after the onset        of symptoms of ischemic stroke.

In an exemplary embodiment the method further comprising the step of

-   -   b) comparing the amount of said cardiac Troponin as determined        in step a to a reference amount, thereby differentiating whether        said subject suffers from cardioembolic stroke or from        non-cardioembolic ischemic stroke.

Thus, the present disclosure, in particular, relates to a method forearly differentiating whether a subject suffers from cardioembolicstroke or from non-cardioembolic ischemic stroke, comprising

-   -   a) determining the amount of a cardiac Troponin in a sample from        a subject suffering from ischemic stroke, obtained immediately        after the onset of symptoms of ischemic stroke, and    -   b) comparing the amount of said cardiac Troponin as determined        in step a) to a reference amount, whereby it is differentiated        whether said subject suffers from cardioembolic ischemic stroke        or from non-cardioembolic ischemic stroke.

According to exemplary embodiments, it is differentiated, whether asubject suffers from cardioembolic ischmemic stroke or fromnon-cardioembolic ischemic stroke by carrying out the further step of c)diagnosing whether a subject suffers from cardioembolic ischemic strokeor from non-cardioembolic ischemic stroke, based on the results of thecomparison carried out in step b).

In an exemplary embodiment of the method of the present disclosure, stepa) further comprises the determination of the amount of a natriureticpeptide in the sample from the subject obtained immediately after theonset of ischemic stroke. Thus, according to exemplary embodiments, thedetermined amount of the natriuretic peptide is compared in step b) to areference amount for a natriuretic peptide.

Thus, the present disclosure also relates to a method for earlydifferentiating whether a subject suffers from cardioembolic stroke orfrom non-cardioembolic ischemic stroke, comprising

-   -   a) determining the amount of a cardiac Troponin and of        natriuretic peptide in a sample from a subject suffering from        ischemic stroke, obtained immediately after the onset of        symptoms of ischemic stroke, and    -   b) comparing the amount of said cardiac Troponin as determined        in step a) to a reference amount for the cardiac Troponin, and        the amount of said natriuretic peptide to a reference amount for        the natriuretic peptide, whereby it is differentiated whether        said subject suffers from cardioembolic stroke or from        non-cardioembolic ischemic stroke.

The method of the present disclosure, may include, is an ex vivo method.Moreover, it may comprise steps in addition to those explicitlymentioned above. For example, further steps may relate to samplepre-treatments or evaluation of the results obtained by the method. Themethod may be carried out manually or assisted by automation.Preferably, step (a) and/or (b) may in total or in part be assisted byautomation, e.g., by a suitable robotic and sensory equipment for thedetermination in step (a) or a computer-implemented comparison and/ordifferentiation based on said comparison in step (b).

Accordingly, the present disclosure also preferably relates to a systemfor early differentiating whether a subject suffers from cardioembolicischemic stroke or from non-cardioembolic ischemic stroke, comprising

-   -   a) an analyzer unit configured to contact, in vitro, a portion        of a sample from a subject who suffers from ischemic stroke with        a ligand comprising specific binding affinity for a cardiac        Troponin,    -   b) an analyzer unit configured to detect a signal from the        portion of the sample from the subject contacted with the        ligand,    -   c) a computing device having a processor and in operable        communication with said analysis units, and    -   d) a non-transient machine readable media including a plurality        of instruction executable by a the processor, the instructions,        when executed calculate an amount of the cardiac Troponin, and        compare the amount of the marker with a reference amount,        thereby differentiating whether a subject suffers from        cardioembolic ischemic stroke or from non-cardioembolic ischemic        stroke.

The term “differentiating” as used herein means to distinguish betweencardioembolic stroke and non-cardioembolic stroke in a patient sufferingfrom ischemic stroke. The term as used herein, preferably, includesdifferentially diagnosing cardioembolic ischemic stroke andnon-cardioembolic ischemic stroke in a subject. As will be understood bythose skilled in the art, such an assessment is usually not intended tobe correct for 100% of the subjects to be differentially diagnosed. Theterm, however, requires that a statistically significant portion ofsubjects can be correctly diagnosed. Whether a diagnosis/differentiationis correct can be confirmed by methods well known in the art. Moreover,whether a portion is statistically significant can be determined withoutfurther ado by the person skilled in the art using various well knownstatistic evaluation tools, e.g., determination of confidence intervals,p-value determination, Student's t-test, Mann-Whitney test etc. Detailsare found in Dowdy and Wearden, Statistics for Research, John Wiley &Sons, New York 1983. Preferred confidence intervals are at least 90%, atleast 95%, at least 97%, at least 98% or at least 99%. The p-values are,preferably, 0.1, 0.05, 0.01, 0.005, or 0.0001.

The term “subject” as used herein relates to animals, preferablymammals, and, more preferably, humans. Preferably, the subject does ornot suffer from acute infections. Moreover, it is further envisaged thatthe subject does not suffer from acute coronary syndrome and/or fromchronic renal failure. In particular, the subject in the context withthe aforementioned method shall have normal kidney function.Furthermore, the subject is, preferably, a subject presenting to anemergency unit.

The definition of the subject given herein, preferably, applies to thesubject to be tested in accordance with the method of the presentdisclosure as well as to the subject/subjects from which the referenceamount is derived.

The subject to be tested in accordance with the method of the presentdisclosure shall suffer from ischemic stroke. The term “ischemic stroke”(herein also referred to as “stroke”) is well known by the skilledperson (see e.g. Adams et al., Guidelines for the Early Management ofAdults With Ischemic Stroke, A Guideline From the American HeartAssociation/American Stroke Association Stroke Council, ClinicalCardiology Council, Cardiovascular Radiology and Intervention Council,and the Atherosclerotic Peripheral Vascular Disease and Quality of CareOutcomes in Research Interdisciplinary Working Groups in Stroke. 2007;38:1655; or Stroke Genetics, edited by Hugh S. Markus, Chapter 1 “Anintroduction to stroke, Oxford University Press, Incorporated, PublishDate 06/03, both of which are herewith incorporated by reference withrespect to its entire disclosure content). As used herein, the term,preferably, refers to cerebral ischemic stroke. Ischemic stroke iscaused by reduced blood flow to the brain or parts thereof which leadsto a reduced delivery (undersupply) of oxygen to brain cells. Ischemicstroke may be characterized by tissue anemia caused by obstruction ofthe inflow of arterial blood. It may lead to irreversible tissue damagedue to brain cell death.

There are various classification systems for ischemic stroke. The OxfordCommunity Stroke Project classification (OCSP, also known as the Bamfordor Oxford classification) relies primarily on the initial symptoms;based on the extent of the symptoms, the stroke episode is classified astotal anterior circulation infarct (TACI), partial anterior circulationinfarct (PACI), lacunar infarct (LACI) or posterior circulation infarct(POCI). These four entities predict the extent of the stroke, the areaof the brain affected, the underlying cause, and the prognosis.

Preferably, the so called TOAST criteria are applied herein. For theTOAST criteria, see e.g. Donnan G A, Fisher M, Macleod M, Davis S M (May2008). “Stroke”. Lancet 371 (9624): 1612-23 or “Classification ofsubtype of acute ischemic stroke. Definitions for use in a multicenterclinical trial. TOAST. Trial of Org 10172 in Acute Stroke Treatment”.Stroke 24 (1): 35-41, both of which are herein incorporated by referencewith respect to the entire disclosure content. The TOAST (Trial of Org10172 in Acute Stroke Treatment) classification is based on clinicalsymptoms as well as results of further investigations; on this basis, astroke is classified as being due to (1) embolism of cardiac origin(cardioembolic stroke) (2) thrombosis or embolism due to atherosclerosisof a large artery (large artery stenosis, atherothrombotic stroke), (3)occlusion of a small blood vessel (lacunar stroke) or (4) undeterminedcause (two possible causes: no cause identified, or incompleteinvestigation). Thus, preferred non-cardioembolic ischemic strokes areatherothrombotic stroke (see 2) and lacunar stroke (see 3).

Whether a subject suffers from stroke, in particular from ischemicstroke can be determined by well-known methods. Moreover, symptoms ofstroke are well known in the art and e.g. described in Adams et al.(loc. cit.). E.g., stroke symptoms include sudden numbness or weaknessof face, arm or leg, especially on one side of the body, suddenconfusion, trouble speaking or understanding, sudden trouble seeing inone or both eyes, and sudden trouble walking, dizziness, loss of balanceor coordination.

The term “sample” refers to a sample of a body fluid, to a sample ofseparated cells or to a sample from a tissue or an organ. Samples ofbody fluids can be obtained by well known techniques and include,preferably, samples of blood, plasma, serum, or urine, more preferably,samples of blood, plasma or serum. Tissue or organ samples may beobtained from any tissue or organ by, e.g., biopsy. Separated cells maybe obtained from the body fluids or the tissues or organs by separatingtechniques such as centrifugation or cell sorting. Preferably, cell-,tissue- or organ samples are obtained from those cells, tissues ororgans which express or produce the peptides referred to herein.

The sample to be tested in the context of the method of the presentdisclosure shall have been obtained immediately after the onset ofsymptoms of stroke (as well as the reference sample). Preferably, asample is deemed to have been obtained immediately after the onset ofsymptoms of stroke if it has been obtained from said subject not morethan 24 hours, in particular not more than 12 hours after the onset ofsymptoms of stroke. More preferably, a sample is deemed to have beenobtained immediately after the onset of symptoms of stroke if it hasbeen obtained from said subject not more than 6 hours, and even morepreferably not more than 3 hours after the onset of symptoms of ischemicstroke. Further envisaged is that the sample has been obtained not morethan one or two hours after the onset of stroke symptoms.

The term “cardiac Troponin” refers to all Troponin isoforms expressed incells of the heart and, preferably, the subendocardial cells. Theseisoforms are well characterized in the art as described, e.g., inAnderson 1995, Circulation Research, vol. 76, no. 4: 681-686 andFerrieres 1998, Clinical Chemistry, 44: 487-493. Preferably, cardiacTroponin refers to Troponin T and/or Troponin I, and, most preferably,to Troponin T. It is to be understood that isoforms of Troponins may bedetermined in the method of the present disclosure together, i.e.simultaneously or sequentially, or individually, i.e. withoutdetermining the other isoform at all. Amino acid sequences for humanTroponin T and human Troponin I are disclosed in Anderson, loc cit andFerrieres 1998, Clinical Chemistry, 44: 487-493.

The term “cardiac Troponin” encompasses also variants of theaforementioned specific Troponins, i.e., preferably, of Troponin I, andmore preferably, of Troponin T. Such variants have at least the sameessential biological and immunological properties as the specificcardiac Troponins. In particular, they share the same essentialbiological and immunological properties if they are detectable by thesame specific assays referred to in this specification, e.g., by ELISAAssays using polyclonal or monoclonal antibodies specificallyrecognizing the said cardiac Troponins. Moreover, it is to be understoodthat a variant as referred to in accordance with the present disclosureshall have an amino acid sequence which differs due to at least oneamino acid substitution, deletion and/or addition wherein the amino acidsequence of the variant is still, preferably, at least about 50%, atleast about 60%, at least about 70%, at least about 80%, at least about85%, at least about 90%, at least about 92%, at least about 95%, atleast about 97%, at least about 98%, or at least about 99% identicalwith the amino sequence of the specific Troponin. Preferably, the degreeof identity is to be determined by comparing two optimally alignedsequences over a comparison window, where the fragment of amino acidsequence in the comparison window may comprise additions or deletions(e.g., gaps or overhangs) as compared to the reference sequence (whichdoes not comprise additions or deletions) for optimal alignment. Thepercentage is calculated by determining the number of positions at whichthe identical amino acid residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison andmultiplying the result by 100 to yield the percentage of sequenceidentity. Optimal alignment of sequences for comparison may be conductedby the local homology algorithm of Smith and Waterman Add. APL. Math.2:482 (1981), by the homology alignment algorithm of Needleman andWunsch J. Mol. Biol. 48:443 (1970), by the search for similarity methodof Pearson and Lipman Proc. Natl. Acad. Sci. (USA) 85: 2444 (1988), bycomputerized implementations of these algorithms (GAP, BESTFIT, BLAST,PASTA, and TFASTA in the Wisconsin Genetics Software Package, GeneticsComputer Group (GCG), 575 Science Dr., Madison, Wis.), or by visualinspection. Given that two sequences have been identified forcomparison, GAP and BESTFIT are preferably employed to determine theiroptimal alignment and, thus, the degree of identity. Preferably, thedefault values of 5.00 for gap weight and 0.30 for gap weight length areused. Variants may be allelic variants or any other species specifichomologs, paralogs, or orthologs. Moreover, the variants referred toherein include fragments of the specific cardiac Troponins or theaforementioned types of variants as long as these fragments have theessential immunological and biological properties as referred to above.Preferably, the cardiac troponin variants have immunological properties(i.e. epitope composition) comparable to those of human troponin T ortroponin I. Thus, the variants shall be recognizable by theaforementioned means or ligands used for determination of theconcentration of the cardiac troponins. Thus, the variants shall berecognizable by the aforementioned means or ligands used fordetermination of the concentration of the cardiac troponins. Suchfragments may be, e.g., degradation products of the Troponins. Furtherincluded are variants which differ due to posttranslationalmodifications such as phosphorylation or myristylation. Preferably thebiological property of troponin I and its variant is the ability toinhibit actomyosin ATPase or to inhibit angiogenesis in vivo and invitro, which may e.g. be detected based on the assay described by Moseset al. 1999 PNAS USA 96 (6): 2645-2650). Preferably the biologicalproperty of troponin T and its variant is the ability to form a complexwith troponin C and I, to bind calcium ions or to bind to tropomyosin,preferably if present as a complex of troponin C, I and T or a complexformed by troponin C, troponin I and a variant of troponin T. It isknown that low concentrations of circulating cardiac troponin may bedetected in subjects at various conditions, but further studies arerequired to understand their respective role and rate (Masson et al.,Curr Heart Fail Rep (2010) 7:15-21).

The term “natriuretic peptide” comprises Atrial Natriuretic Peptide(ANP)-type and Brain Natriuretic Peptide (BNP)-type peptides andvariants thereof having the same predictive potential. Natriureticpeptides according to the present disclosure comprise ANP-type andBNP-type peptides and variants thereof (see e.g. Bonow, 1996,Circulation 93: 1946-1950). ANP-type peptides comprise pre-proANP,proANP, NT-proANP, and ANP. BNP-type peptides comprise pre-proBNP,proBNP, NT-proBNP, and BNP. The pre-pro peptide (134 amino acids in thecase of pre-proBNP) comprises a short signal peptide, which isenzymatically cleaved off to release the pro peptide (108 amino acids inthe case of proBNP). The pro peptide is further cleaved into anN-terminal pro peptide (NT-pro peptide, 76 amino acids in case ofNT-proBNP) and the active hormone (32 amino acids in the case of BNP, 28amino acids in the case of ANP). Preferably, natriuretic peptidesaccording to the present disclosure are NT-proANP, ANP, and, morepreferably, NT-proBNP, BNP, and variants thereof. ANP and BNP are theactive hormones and have a shorter half-life than their respectiveinactive counterparts, NT-proANP and NT-proBNP. BNP is metabolized inthe blood, whereas NT-proBNP circulates in the blood as an intactmolecule and as such is eliminated renally. The in-vivo half-life ofNT-proBNP is 120 min longer than that of BNP, which is 20 min (Smith2000, J Endocrinol. 167: 239-46.). Preanalytics are more robust withNT-proBNP allowing easy transportation of the sample to a centrallaboratory (Mueller 2004, Clin Chem Lab Med 42: 942-4.). Blood samplescan be stored at room temperature for several days or may be mailed orshipped without recovery loss. In contrast, storage of BNP for 48 hoursat room temperature or at 4° Celsius leads to a concentration loss of atleast 20% (Mueller loc.cit.; Wu 2004, Clin Chem 50: 867-73.). Therefore,depending on the time-course or properties of interest, eithermeasurement of the active or the inactive forms of the natriureticpeptide can be advantageous. The most preferred natriuretic peptidesaccording to the present disclosure are NT-proBNP or variants thereof.As briefly discussed above, the human NT-proBNP, as referred to inaccordance with the present disclosure, is a polypeptide comprising,preferably, 76 amino acids in length corresponding to the N-terminalportion of the human NT-proBNP molecule. The structure of the human BNPand NT-proBNP has been described already in detail in the prior art,e.g., WO 02/089657, WO 02/083913 or Bonow loc. cit. Preferably, humanNT-proBNP as used herein is human NT-proBNP as disclosed in EP 0 648 228B1. These prior art documents are herewith incorporated by referencewith respect to the specific sequences of NT-proBNP and variants thereofdisclosed therein. The NT-proBNP referred to in accordance with thepresent disclosure further encompasses allelic and other variants ofsaid specific sequence for human NT-proBNP discussed above.Specifically, envisaged are variant polypeptides which are on the aminoacid level preferably, at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%,97%, 98%, or 99% identical to human NT-proBNP, preferably over theentire length of human NT-proBNP. The degree of identity between twoamino acid sequences can be determined by algorithms well known in theart. Preferably, the degree of identity is to be determined by comparingtwo optimally aligned sequences over a comparison window, where thefragment of amino acid sequence in the comparison window may compriseadditions or deletions (e.g., gaps or overhangs) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment. The percentage is calculated by determining thenumber of positions at which the identical amino acid residue occurs inboth sequences to yield the number of matched positions, dividing thenumber of matched positions by the total number of positions in thewindow of comparison and multiplying the result by 100 to yield thepercentage of sequence identity. Optimal alignment of sequences forcomparison may be conducted by the local homology algorithm of Smith andWaterman Add. APL. Math. 2:482 (1981), by the homology alignmentalgorithm of Needleman and Wunsch J. Mol. Biol. 48:443 (1970), by thesearch for similarity method of Pearson and Lipman Proc. Natl. Acad.Sci. (USA) 85: 2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, BLAST, PASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group (GCG), 575 ScienceDr., Madison, Wis.), or by visual inspection. Given that two sequenceshave been identified for comparison, GAP and BESTFIT are preferablyemployed to determine their optimal alignment and, thus, the degree ofidentity. Preferably, the default values of 5.00 for gap weight and 0.30for gap weight length are used. Variants referred to above may beallelic variants or any other species specific homologs, paralogs, ororthologs. Substantially similar and also envisaged are proteolyticdegradation products which are still recognized by the diagnostic meansor by ligands directed against the respective full-length peptide. Alsoencompassed are variant polypeptides having amino acid deletions,substitutions, and/or additions compared to the amino acid sequence ofhuman NT-proBNP as long as the said polypeptides have NT-proBNPproperties. NT-proBNP properties as referred to herein are immunologicaland/or biological properties. Preferably, the NT-proBNP variants haveimmunological properties (i.e. epitope composition) comparable to thoseof human NT-proBNP. Thus, the variants shall be recognizable by theaforementioned means or ligands used for determination of the amount ofthe natriuretic peptides. Biological and/or immunological NT-proBNPproperties can be detected by the assay described in Karl et al. (Karl1999, Scand J Clin Lab Invest 230:177-181), Yeo et al. (Yeo 2003,Clinica Chimica Acta 338:107-115). Variants also includeposttranslationally modified peptides such as glycosylated peptides.Further, a variant in accordance with the present disclosure is also apeptide or polypeptide which has been modified after collection of thesample, for example by covalent or non-covalent attachment of a label,particularly a radioactive or fluorescent label, to the peptide.

Determining the amount of a peptide or polypeptide referred to in thisspecification relates to measuring the amount or concentration,preferably, semi-quantitatively or quantitatively. Measuring can be donedirectly or indirectly. Direct measuring relates to measuring the amountor concentration of the peptide or polypeptide based on a signal whichis obtained from the peptide or polypeptide itself and the intensity ofwhich directly correlates with the number of molecules of the peptidepresent in the sample. Such a signal—sometimes referred to herein asintensity signal—may be obtained, e.g., by measuring an intensity valueof a specific physical or chemical property of the peptide orpolypeptide. Indirect measuring includes measuring of a signal obtainedfrom a secondary component (i.e. a component not being the peptide orpolypeptide itself) or a biological read out system, e.g., measurablecellular responses, ligands, labels, or enzymatic reaction products.

In accordance with the present disclosure, determining the amount of apeptide or polypeptide can be achieved by all known means fordetermining the amount of a peptide in a sample. Said means compriseimmunoassay and methods which may utilize labeled molecules in varioussandwich, competition, or other assay formats. Such assays are,preferably, based on detection agents such as antibodies whichspecifically recognize the peptide or polypeptide to be determined. Thedetection agents shall be either directly or indirectly capable ofgenerating a signal indicating the presence or absence of the peptide orpolypeptide. Moreover, the signal strength can, preferably, becorrelated directly or indirectly (e.g. reverse-proportional) to theamount of polypeptide present in a sample. Further suitable methodscomprise measuring a physical or chemical property specific for thepeptide or polypeptide such as its precise molecular mass or NMRspectrum. Said methods comprise, preferably, biosensors, optical devicescoupled to immunoassays, biochips, analytical devices such asmass-spectrometers, NMR-analyzers, or chromatography devices. Further,methods include micro-plate ELISA-based methods, fully-automated orrobotic immunoassays (available for example on Elecsys™ analyzers), CBA(an enzymatic Cobalt Binding Assay, available for example onRoche-Hitachi™ analyzers), and latex agglutination assays (available forexample on Roche-Hitachi™ analyzers).

Preferably, determining the amount of a peptide or polypeptide comprisesthe steps of (a) contacting a cell capable of eliciting a cellularresponse the intensity of which is indicative of the amount of thepeptide or polypeptide with the said peptide or polypeptide for anadequate period of time, (b) measuring the cellular response. Formeasuring cellular responses, the sample or processed sample is,preferably, added to a cell culture and an internal or external cellularresponse is measured. The cellular response may include the measurableexpression of a reporter gene or the secretion of a substance, e.g. apeptide, polypeptide, or a small molecule. The expression or substanceshall generate an intensity signal which correlates to the amount of thepeptide or polypeptide.

Also preferably, determining the amount of a peptide or polypeptidecomprises the step of measuring a specific intensity signal obtainablefrom the peptide or polypeptide in the sample. As described above, sucha signal may be the signal intensity observed at an m/z variablespecific for the peptide or polypeptide observed in mass spectra or aNMR spectrum specific for the peptide or polypeptide.

Determining the amount of a peptide or polypeptide may, preferably,comprises the steps of (a) contacting the peptide with a specificligand, (b) (optionally) removing non-bound ligand, (c) measuring theamount of bound ligand.

According to a preferred embodiment, said steps of contacting, removingand measuring may be performed by an analyzer unit of the systemdisclosed herein. According to some embodiments, said steps may beperformed by a single analyzer unit of said system or by more than oneanalyzer unit in operable communication with each other. For example,according to a specific embodiment, said system disclosed herein mayinclude a first analyzer unit for performing said steps of contactingand removing and a second analyzer unit, operably connected to saidfirst analyzer unit by a transport unit (for example, a robotic arm),which performs said step of measuring.

The bound ligand, in particular the ligand or the ligand/peptidecomplex, will generate an intensity signal. Binding according to thepresent disclosure includes both covalent and non-covalent binding. Aligand according to the present disclosure can be any compound, e.g., apeptide, polypeptide, nucleic acid, or small molecule, binding to thepeptide or polypeptide described herein. Preferred ligands includeantibodies, nucleic acids, peptides or polypeptides such as receptors orbinding partners for the peptide or polypeptide and fragments thereofcomprising the binding domains for the peptides, and aptamers, e.g.nucleic acid or peptide aptamers. Methods to prepare such ligands arewell-known in the art. For example, identification and production ofsuitable antibodies or aptamers is also offered by commercial suppliers.The person skilled in the art is familiar with methods to developderivatives of such ligands with higher affinity or specificity. Forexample, random mutations can be introduced into the nucleic acids,peptides or polypeptides. These derivatives can then be tested forbinding according to screening procedures known in the art, e.g. phagedisplay. Antibodies as referred to herein include both polyclonal andmonoclonal antibodies, as well as fragments thereof, such as Fv, Fab andF(ab)2 fragments that are capable of binding antigen or hapten. Thepresent disclosure also includes single chain antibodies and humanizedhybrid antibodies wherein amino acid sequences of a non-human donorantibody exhibiting a desired antigen-specificity are combined withsequences of a human acceptor antibody. The donor sequences will usuallyinclude at least the antigen-binding amino acid residues of the donorbut may comprise other structurally and/or functionally relevant aminoacid residues of the donor antibody as well. Such hybrids can beprepared by several methods well known in the art. Preferably, theligand or agent binds specifically to the peptide or polypeptide.Specific binding according to the present disclosure means that theligand or agent should not bind substantially to (“cross-react” with)another peptide, polypeptide or substance present in the sample to beanalyzed. Preferably, the specifically bound peptide or polypeptideshould be bound with at least 3 times higher, more preferably at least10 times higher and even more preferably at least 50 times higheraffinity than any other relevant peptide or polypeptide. Non-specificbinding may be tolerable, if it can still be distinguished and measuredunequivocally, e.g. according to its size on a Western Blot, or by itsrelatively higher abundance in the sample. Binding of the ligand can bemeasured by any method known in the art. Preferably, said method issemi-quantitative or quantitative. Further suitable techniques for thedetermination of a polypeptide or peptide are described in thefollowing.

First, binding of a ligand may be measured directly, e.g. by NMR orsurface plasmon resonance. Measurement of the binding of a ligand,according to preferred embodiments, is performed by an analyzer unit ofa system disclosed herein. Thereafter, an amount of the measured bindingmay be calculated by a computing device of a system disclosed herein.Second, if the ligand also serves as a substrate of an enzymaticactivity of the peptide or polypeptide of interest, an enzymaticreaction product may be measured (e.g. the amount of a protease can bemeasured by measuring the amount of cleaved substrate, e.g. on a WesternBlot). Alternatively, the ligand may exhibit enzymatic properties itselfand the “ligand/peptide or polypeptide” complex or the ligand which wasbound by the peptide or polypeptide, respectively, may be contacted witha suitable substrate allowing detection by the generation of anintensity signal. For measurement of enzymatic reaction products,preferably the amount of substrate is saturating. The substrate may alsobe labeled with a detectable lable prior to the reaction. Preferably,the sample is contacted with the substrate for an adequate period oftime. An adequate period of time refers to the time necessary for andetectable, preferably measurable, amount of product to be produced.Instead of measuring the amount of product, the time necessary forappearance of a given (e.g. detectable) amount of product can bemeasured. Third, the ligand may be coupled covalently or non-covalentlyto a label allowing detection and measurement of the ligand. Labelingmay be done by direct or indirect methods. Direct labeling involvescoupling of the label directly (covalently or non-covalently) to theligand. Indirect labeling involves binding (covalently ornon-covalently) of a secondary ligand to the first ligand. The secondaryligand should specifically bind to the first ligand. Said secondaryligand may be coupled with a suitable label and/or be the target(receptor) of tertiary ligand binding to the secondary ligand. The useof secondary, tertiary or even higher order ligands is often used toincrease the signal. Suitable secondary and higher order ligands mayinclude antibodies, secondary antibodies, and the well-knownstreptavidin-biotin system (Vector Laboratories, Inc.). The ligand orsubstrate may also be “tagged” with one or more tags as known in theart. Such tags may then be targets for higher order ligands. Suitabletags include biotin, digoxygenin, His-Tag, Glutathion-S-Transferase,FLAG, GFP, myc-tag, influenza A virus haemagglutinin (HA), maltosebinding protein, and the like. In the case of a peptide or polypeptide,the tag is preferably at the N-terminus and/or C-terminus. Suitablelabels are any labels detectable by an appropriate detection method.Typical labels include gold particles, latex beads, acridan ester,luminol, ruthenium, enzymatically active labels, radioactive labels,magnetic labels (“e.g. magnetic beads”, including paramagnetic andsuperparamagnetic labels), and fluorescent labels. Enzymatically activelabels include e.g. horseradish peroxidase, alkaline phosphatase,beta-Galactosidase, Luciferase, and derivatives thereof. Suitablesubstrates for detection include di-amino-benzidine (DAB),3,3′-5,5′-tetramethylbenzidine, NBT-BCIP (4-nitro blue tetrazoliumchloride and 5-bromo-4-chloro-3-indolyl-phosphate, available asready-made stock solution from Roche Diagnostics), CDP-Star™ (AmershamBiosciences), ECF™ (Amersham Biosciences). A suitable enzyme-substratecombination may result in a colored reaction product, fluorescence orchemoluminescence, which can be measured according to methods known inthe art (e.g. using a light-sensitive film or a suitable camera system).As for measuring the enyzmatic reaction, the criteria given above applyanalogously. Typical fluorescent labels include fluorescent proteins(such as GFP and its derivatives), Cy3, Cy5, Texas Red, Fluorescein, andthe Alexa dyes (e.g. Alexa 568). Further fluorescent labels areavailable e.g. from Molecular Probes (Oregon). Also the use of quantumdots as fluorescent labels is contemplated. Typical radioactive labelsinclude 35S, 125I, 32P, 33P and the like. A radioactive label can bedetected by any method known and appropriate, e.g. a light-sensitivefilm or a phosphor imager. Suitable measurement methods according thepresent disclosure also include precipitation (particularlyimmunoprecipitation), electrochemiluminescence (electro-generatedchemiluminescence), RIA (radioimmunoassay), ELISA (enzyme-linkedimmunosorbent assay), sandwich enzyme immune tests,electrochemiluminescence sandwich immunoassays (ECLIA),dissociation-enhanced lanthanide fluoro immuno assay (DELFIA),scintillation proximity assay (SPA), turbidimetry, nephelometry,latex-enhanced turbidimetry or nephelometry, or solid phase immunetests. Further methods known in the art (such as gel electrophoresis, 2Dgel electrophoresis, SDS polyacrylamid gel electrophoresis (SDS-PAGE),Western Blotting, and mass spectrometry), can be used alone or incombination with labeling or other detection methods as described above.

The amount of a peptide or polypeptide may be, also preferably,determined as follows: (a) contacting a solid support comprising aligand for the peptide or polypeptide as specified above with a samplecomprising the peptide or polypeptide and (b) measuring the amountpeptide or polypeptide which is bound to the support. The ligand,preferably chosen from the group consisting of nucleic acids, peptides,polypeptides, antibodies and aptamers, is preferably present on a solidsupport in immobilized form. Materials for manufacturing solid supportsare well known in the art and include, inter alia, commerciallyavailable column materials, polystyrene beads, latex beads, magneticbeads, colloid metal particles, glass and/or silicon chips and surfaces,nitrocellulose strips, membranes, sheets, duracytes, wells and walls ofreaction trays, plastic tubes etc. The ligand or agent may be bound tomany different carriers. Examples of well-known carriers include glass,polystyrene, polyvinyl chloride, polypropylene, polyethylene,polycarbonate, dextran, nylon, amyloses, natural and modifiedcelluloses, polyacrylamides, agaroses, and magnetite. The nature of thecarrier can be either soluble or insoluble for the purposes of thedisclosure. Suitable methods for fixing/immobilizing said ligand arewell known and include, but are not limited to ionic, hydrophobic,covalent interactions and the like. It is also contemplated to use“suspension arrays” as arrays according to the present disclosure (Nolan2002, Trends Biotechnol. 20(1):9-12). In such suspension arrays, thecarrier, e.g. a microbead or microsphere, is present in suspension. Thearray consists of different microbeads or microspheres, possiblylabeled, carrying different ligands. Methods of producing such arrays,for example based on solid-phase chemistry and photo-labile protectivegroups, are generally known (U.S. Pat. No. 5,744,305).

The term “amount” as used herein encompasses the absolute amount of apolypeptide or peptide, the relative amount or concentration of the saidpolypeptide or peptide as well as any value or parameter whichcorrelates thereto or can be derived therefrom. Such values orparameters comprise intensity signal values from all specific physicalor chemical properties obtained from the said peptides by directmeasurements, e.g., intensity values in mass spectra or NMR spectra.Moreover, encompassed are all values or parameters which are obtained byindirect measurements specified elsewhere in this description, e.g.,response amounts determined from biological read out systems in responseto the peptides or intensity signals obtained from specifically boundligands. It is to be understood that values correlating to theaforementioned amounts or parameters can also be obtained by allstandard mathematical operations. According to preferred embodiments ofthe subject disclosure, the determination of an “amount” is performed bythe disclosed system, whereby a computing device determines the “amount”based on contacting and measuring steps performed by one or moreanalyzer units of said system.

The term “comparing” as used herein encompasses comparing the amount ofthe peptide or polypeptide comprised by the sample to be analyzed withan amount of a suitable reference source specified elsewhere in thisdescription. It is to be understood that comparing as used herein refersto a comparison of corresponding parameters or values, e.g., an absoluteamount is compared to an absolute reference amount while a concentrationis compared to a reference concentration or an intensity signal obtainedfrom a test sample is compared to the same type of intensity signal of areference sample. The comparison referred to in step (b) of the methodof the present disclosure may be carried out manually or computerassisted. Thus, the comparison referred to in step (b) of the method ofthe present disclosure may be carried out by a computing device (e.g.,of a system disclosed herein). The value of the amount and the referencecan be, e.g., compared to each other and the said comparison can beautomatically carried out by a computer program executing an algorithmfor the comparison. The computer program carrying out the saidevaluation will provide the desired assessment in a suitable outputformat. For a computer assisted comparison, the value of the determinedamount may be compared to values corresponding to suitable referenceswhich are stored in a database by a computer program. The computerprogram may further evaluate the result of the comparison, i.e.automatically provide the desired assessment in a suitable outputformat.

For a computer assisted comparison, the value of the determined amountmay be compared to values corresponding to suitable references which arestored in a database by a computer program. The computer program mayfurther evaluate the result of the comparison, i.e. automaticallyprovides the desired assessment in a suitable output format. The saidresult may, preferably, serve as an aid in differentiating betweencardioembolic and non-cardioembolic ischemic stroke.

For example, a result of a comparison may be given as raw data (absoluteor relative amounts), and in some cases as an indicator in the form of aword, phrase, symbol, or numerical value which may be indicative of aparticular diagnosis.

The term “reference amount” as used herein refers to an amount whichallows for allocation of a subject into either the group of subjectssuffering from cardioembolic ischemic stroke or into a group of subjectsuffering from non-cardioembolic ischemic stroke. Such a referenceamount can be a threshold amount which separates these groups from eachother. Accordingly, the reference amount for a the biomarker Troponinshall be an amount which allows for allocation of a subject into a groupof subjects suffering from cardioembolic ischemic stroke or into a groupof subjects suffering from non-cardioembolic ischemic stroke. A suitablethreshold amount separating the two groups can be calculated withoutfurther ado by the statistical tests referred to herein elsewhere basedon amounts of a cardiac Troponin from either a subject or group ofsubjects suffering from cardioembolic ischemic stroke or a subject orgroup of subjects suffering from non-cardioembolic ischemic stroke.Preferred referenced amounts which can be derived from theaforementioned subjects or group of subjects are indicated elsewhereherein.

Reference amounts can, in principle, be calculated for a cohort ofsubjects as specified above based on the average or mean values for agiven biomarker by applying standard statistically methods. Inparticular, accuracy of a test such as a method aiming to diagnose anevent, or not, is best described by its receiver-operatingcharacteristics (ROC) (see especially Zweig 1993, Clin. Chem.39:561-577). The ROC graph is a plot of all of thesensitivity/specificity pairs resulting from continuously varying thedecision threshold over the entire range of data observed. The clinicalperformance of a diagnostic method depends on its accuracy, i.e. itsability to correctly allocate subjects to a certain prognosis ordiagnosis. The ROC plot indicates the overlap between the twodistributions by plotting the sensitivity versus 1-specificity for thecomplete range of thresholds suitable for making a distinction. On they-axis is sensitivity, or the true-positive fraction, which is definedas the ratio of number of true-positive test results to the product ofnumber of true-positive and number of false-negative test results. Thishas also been referred to as positivity in the presence of a disease orcondition. It is calculated solely from the affected subgroup. On thex-axis is the false-positive fraction, or 1-specificity, which isdefined as the ratio of number of false-positive results to the productof number of true-negative and number of false-positive results. It isan index of specificity and is calculated entirely from the unaffectedsubgroup. Because the true- and false-positive fractions are calculatedentirely separately, by using the test results from two differentsubgroups, the ROC plot is independent of the prevalence of the event inthe cohort. Each point on the ROC plot represents asensitivity/-specificity pair corresponding to a particular decisionthreshold. A test with perfect discrimination (no overlap in the twodistributions of results) has an ROC plot that passes through the upperleft corner, where the true-positive fraction is 1.0, or 100% (perfectsensitivity), and the false-positive fraction is 0 (perfectspecificity). The theoretical plot for a test with no discrimination(identical distributions of results for the two groups) is a 45°diagonal line from the lower left corner to the upper right corner. Mostplots fall in between these two extremes. If the ROC plot fallscompletely below the 45° diagonal, this is easily remedied by reversingthe criterion for “positivity” from “greater than” to “less than” orvice versa. Qualitatively, the closer the plot is to the upper leftcorner, the higher the overall accuracy of the test. Dependent on adesired confidence interval, a threshold can be derived from the ROCcurve allowing for the diagnosis or prediction for a given event with aproper balance of sensitivity and specificity, respectively.Accordingly, the reference to be used for the aforementioned method ofthe present disclosure can be, preferably, a threshold or cut off amountand can be generated, preferably, by establishing a ROC for said cohortas described above and deriving a threshold amount therefrom. Dependenton a desired sensitivity and specificity for a diagnostic method, theROC plot allows deriving suitable thresholds.

The diagnosis/differentiation referred to herein may be provided by thecomputing device of a system disclosed herein based on said comparisonof the calculated “amount” to a reference or a threshold. For example, acomputing device of a system may provide an indicator, in the form of aword, symbol, or numerical value which is indicative of onecardioembolic stroke or non-cardioembolic stroke.

Preferably, the reference amount(s) is (are) derived from a subject or agroup of subjects known to suffer from cardioembolic ischemic stroke. Inthis case, an essentially identical amount or an increased amount of acardiac Troponin in the test sample as compared to reference amount is,preferably, indicative for cardioembolic ischemic stroke. If also anatriuretic peptide is determined, an essentially identical amount or anincreased amount of a cardiac Troponin and of the natriuretic peptide inthe test sample as compared to reference amount for the cardiac Troponinand the reference amount for the natriuretic peptide is, preferably,indicative for cardioembolic ischemic stroke.

Also preferably, the reference amount for a cardiac Troponin (and,optionally, of the natriuretic peptide) may be derived from a subject ora group of subjects known to suffer from non-cardioembolic ischemicstroke. In this case, an essentially identical amount or a decreasedamount of the cardiac Troponin (and, optionally, of the natriureticpeptide) in the test sample as compared to reference amount isindicative for non-cardioembolic ischemic stroke. If also a natriureticpeptide is determined, an essentially identical amount or a decreasedamount of the cardiac Troponin of the natriuretic peptide in the testsample as compared to the reference amount for the cardiac Troponin andthe reference amount for the natriuretic peptide is indicative fornon-cardioembolic ischemic stroke.

The reference amount applicable for an individual subject may varydepending on various physiological parameters such as age, gender, orsubpopulation, as well as on the means used for the determination of thepolypeptide or peptide referred to herein. A suitable reference amountmay be determined from a reference sample to be analyzed together, i.e.simultaneously or subsequently, with the test sample.

Further, the reference amount may define a threshold amount, inparticular a calculated reference amount, for the cardiac Troponin (and,optionally for the natriuretic peptide), whereby an amount of Troponin(and, optionally of the natriuretic peptide) in the sample of the testsubject larger than the respective threshold shall be indicative forcardioembolic ischemic stroke, while an amount of Troponin (and,optionally, of the natriuretic peptide) in the sample of the testsubject lower than the respective threshold shall be indicative fornon-cardioembolic stroke.

Preferred reference amounts are indicated herein below.

A preferred reference amount indicating cardioembolic ischemic stroke isan amount of a cardiac troponin, in particular of Troponin T of about 8pg/ml to about 40 pg/ml and, more preferably, 10 to 30 pg/ml, about 11.6to about 20 pg/ml, even more preferably of about 15 to 20 pg/ml. Evenmore preferably, the reference is an amount of about 8, 10, or 11.6pg/ml. A test amount being essentially identical or increased shall beindicative for cardioembolic ischemic stroke while a decreased amount inthe test sample as compared to the reference amount shall be indicativefor non-cardioembolic ischemic stroke. Preferably, the aforementionedreference amounts are derived from a subject or a group of subjectsknown to suffer from cardioembolic ischemic stroke.

The present disclosure is, particularly, useful for ruling outcardioembolic stroke. In particular, a test amount of a cardiacTroponin, preferably, of Troponin T, being lower than 5 pg/ml, inparticular lower than 3 or lower than 2 pg/mg indicates that the subjectdoes not suffer from cardioembolic stroke (and, thus, preferably,suffers from non-cardioembolic stroke).

A preferred reference amount indicating cardioembolic ischemic stroke isan amount of a natriuretic peptide, in particular, of NT-proBNP of about500 pg/ml to about 1500 pg/ml and, more preferably, 700 to 1300 pg/ml,even more preferably of about 800 to 1000 pg/ml. Even more preferably,the reference is an amount of about 700, 800, or most preferably 900pg/ml. A test amount being essentially identical or increased shall beindicative for cardioembolic ischemic stroke while a decreased amount inthe test sample as compared to the reference amount shall be indicativefor non-cardioembolic ischemic stroke. Preferably, the aforementionedreference amounts are derived from a subject or a group of subjectsknown to suffer from cardioembolic ischemic stroke (in addition to theamount of the cardiac Troponin).

As set forth above, the present disclosure is, particularly, useful forruling out cardioembolic stroke. In particular, a test amount of anatriuretic peptide, in particular of NT-proBNP being lower than 250pg/ml, in particular lower than 200 or lower than 150 pg/mg indicatesthat the subject does not suffer from cardioembolic stroke (and, thus,preferably, suffers from non-cardioembolic stroke).

The term “about” in the context of the present disclosure means +/−20%,+/−10%, +/−5%, +/−2% or +/−1% from the said values. This also takes intoaccount usual deviations caused by measurement techniques, statisticsand the like.

In some embodiments of the method of the present disclosure, said methodfurther comprises recommending a therapy for said subject, inparticular, if the subject has been diagnosed to suffer fromcardioembolic stroke. A therapy that can be recommended in a subject whosuffers from cardioembolic stroke is lytic therapy and/oranticoagulation therapy (see e.g. Cairns J. A. et al Canadian J ofCardiology 2011: 27: 74-90 or Camm A. J. et al Eur Heart Journal 2010:31: 2369-429 which are both incorporated herein by reference).

In an aspect of the disclosure, a method for differentiating whether asubject suffers from cardioembolic ischemic stroke or fromnon-cardioembolic ischemic stroke, is contemplated, said methodcomprising:

-   -   a) determining the amount of a cardiac Troponin in a sample from        a subject suffering from ischemic stroke by (i) bringing the        sample into contact with a detection agent that specifically        binds to said cardiac Troponin for a time sufficient to allow        for the formation of a complex of the said detection agent and        the cardiac Troponin from the sample, (ii) measuring the amount        of the formed complex, wherein the said amount of the formed        complex is proportional to the amount of the at cardiac Troponin        present in the sample, and (iii) transforming the amount of the        formed complex into an amount of the cardiac Troponin reflecting        the amount of the marker present in the sample;    -   b) comparing said amount to a reference; and    -   c) establishing an aid for differentiating whether a subject        suffers from cardioembolic ischemic stroke or from        non-cardioembolic ischemic stroke, based on the result of the        comparison made in step b).

Preferably, also the amount of a natriuretic peptide is determined.

In another aspect of the disclosure, a system for differentiatingwhether a subject suffers from cardioembolic ischemic stroke or fromnon-cardioembolic ischemic stroke, is contemplated, comprising:

-   -   a) an analyzer unit configured to bringing a sample from a        subject who suffers from ischemic stroke into contact with a        detection agent that specifically binds to the marker cardiac        Troponin for a time sufficient to allow for the formation of a        complex of the said detection agent and the marker from the        sample,    -   b) an analyzer unit configured to measure the amount of the        formed complex, wherein the said amount of the formed complex is        proportional to the amount of the marker present in the sample,    -   c) a computing device having a processor and in operable        communication with said analysis units, and    -   d) a non-transient machine readable media including a plurality        of instructions executable by the processor, the instructions,        when executed transform the amount of the formed complex into an        amount of the marker reflecting the amount of the marker present        in the sample, compare said amount to a reference, and establish        an aid for differentiating whether a subject suffers from        cardioembolic ischemic stroke or from non-cardioembolic ischemic        stroke based on the result of said comparison to said reference.

Preferably, the system also comprises a detection agent thatspecifically binds to natriuretic peptide.

A suitable detection agent may be, in an aspect, an antibody which isspecifically binds to the cardiac troponin, in a sample of a subject tobe investigated by the method of the disclosure. Another detection agentthat can be applied, in an aspect, may be an aptamere which specificallybinds to the marker in the sample. In yet an aspect the, sample isremoved from the complex formed between the detection agent and themarker prior to the measurement of the amount of formed complex.Accordingly, in an aspect, the detection agent may be immobilized on asolid support. In yet an aspect, the sample can be removed from theformed complex on the solid support by applying a washing solution. Theformed complex shall be proportional to the amount of the marker presentin the sample. It will be understood that the specificity and/orsensitivity of the detection agent to be applied defines the degree ofproportion of at least one marker comprised in the sample which iscapable of being specifically bound. Further details on how thedetermination can be carried out are also found elsewhere herein. Theamount of formed complex shall be transformed into an amount of themarker reflecting the amount indeed present in the sample. Such anamount, in an aspect, may be essentially the amount present in thesample or may be, in another aspect, an amount which is a certainproportion thereof due to the relationship between the formed complexand the amount present in the original sample.

In yet an aspect of the aforementioned method, step a) may be carriedout by an analyzer unit, in an aspect, an analyzer unit as definedelsewhere herein.

In an aspect of the method of the disclosure, the amount determined instep a) is compared to a reference. In an aspect, the reference is areference as defined elsewhere herein. In yet another aspect, thereference takes into account the proportional relationship between themeasured amount of complex and the amount present in the originalsample. Thus, the references applied in an aspect of the method of thedisclosure are artificial references which are adopted to reflect thelimitations of the detection agent that has been used. In anotheraspect, said relationship can be also taken into account when carryingout the comparison, e.g., by including a normalization and/or correctioncalculation step for the determined amount prior to actually comparingthe value of the determined amount and the reference. Again, thenormalization and/or correction calculation step for the determinedamount adopts the comparison step such that the limitations of thedetection agent that has been used are reflected properly. In an aspect,the comparison is carried out automatically, e.g., assisted by acomputer system or the like.

The aid for differentiating whether a subject suffers from cardioembolicischemic stroke or from non-cardioembolic ischemic stroke is establishedbased on the comparison carried out in step b) by allocating the subjecteither (i) the group of subjects who suffer from cardioembolic stroke or(ii) the group of who suffer from non-cardioembolic stroke as set forthherein elsewhere. As discussed elsewhere herein already, the allocationof the investigated subject must not be correct in 100% of theinvestigated cases. Moreover, the groups of subjects into which theinvestigated subject is allocated are artificial groups in that they areestablished based on statistical considerations, i.e. a certainpreselected degree of likelihood based on which the method of thedisclosure shall operate. In an aspect of the disclosure, the aid forthe differentiation is established automatically, e.g., assisted by acomputing device or the like, as described and disclosed herein.

In an aspect of the method of the disclosure, said method furthercomprises a step of recommending and/or managing the subject accordingto the result established in step c) as set forth elsewhere herein indetail.

In an aspect of the aforementioned method, steps b) and/or c) arecarried out by one or more analyzer units as set forth elsewhere herein.

Method for Diagnosing Atrial Fibrillation

Atrial fibrillation (AF) is frequently no recognised by the patient.This is the case in approximately 40% of patients indicating thathistory taking in insensitive for the diagnosis of atrial fibrillation(Kamel H. et al, Curr Atheroscler Rep 2011: 13: 338-343). While thesenumbers relate to persistent atrial fibrillation, paroxysmal atrialfibrillation is even more difficult to diagnose and can only be capturedby inpatient cardiac telemetry or even Jolter Monitoring. The latterhaving the advantage, that the ECG is recorded and can later on bereviewed by an experienced physician. Using Holter ECGparoxysmal—previously unrecognized atrial fibrillation was more frequentthan persistent atrial fribillation (Rizos T. et all. Cerebrovasc. Dis2011: 32: 276-282). In order to capture paroxysmal/ischmemic atrialfibrillation reliably 72 h Holter monitoring appears necessary(Gumbinger C et al, Europ. J of Neurology 2011 ahead of publication).Thus recognition of paroxysmal atrial fibrillation is a significantchallenge, specifically as at least 1% of the general population haspersistent atrial fibrillation and the frequency increases with age(Rizos T. et al).

The inventors have found that the determination of a cardiac Troponinallows for diagnosing atrial fibrillation. Interestingly, patients withintermittent AF had also increased cardiac Troponin levels. Therefore,also patients exhibiting intermittent AF can be identified bydetermining the amount of cardiac Troponin.

The definitions and explanations given herein above apply mutatismutandis to the following embodiments of the present disclosure.

Moreover, the present disclosure relates to a method for diagnosingatrial fibrillation in a subject suspected to suffer from atrialfibrillation, comprising a) determining the amount of a cardiac Troponinin a sample from said subject.

In a preferred embodiment the method comprises the further step of

-   -   b) comparing the, thus, determined amount of said cardiac        Troponin to a reference amount. Thereby, intermittent atrial        fibrillation is diagnosed.

Accordingly, the present disclosure relates to a method for diagnosingatrial fibrillation in a subject suspected to suffer from atrialfibrillation, comprising

-   -   a) determining the amount of a cardiac Troponin in a sample from        said subject, and    -   b) comparing the, thus, determined amount of said cardiac        Troponin to a reference amount, whereby atrial fibrillation is        diagnosed.

Preferably, it is diagnosed whether a subject suffers from atrialfibrillation, or not, by carrying out the further step c) of diagnosingwhether the subject suffers from atrial fibrillation, or not.

Preferably, intermittent atrial fibrillation is diagnosed.

Accordingly, the present disclosure, in particular relates to a methodfor diagnosing intermittent atrial fibrillation in a subject suspectedto suffer from atrial fibrillation, comprising

-   -   a) determining the amount of a cardiac Troponin in a sample from        said subject, and    -   b) comparing the, thus, determined amount of said cardiac        Troponin to a reference amount, whereby intermittent atrial        fibrillation is diagnosed.

In a preferred embodiment of the method of the present disclosure, stepa) further comprises the determination of the amount of a natriureticpeptide in the sample from the subject. Preferably, the, thus,determined amount of the natriuretic peptide is compared in step b) to areference amount for a natriuretic peptide.

Thus, the present disclosure also relates to a method for diagnosingatrial fibrillation in a subject suspected to suffer from atrialfibrillation, comprising

-   -   a) determining the amount of a cardiac Troponin and of        natriuretic peptide in a sample from said subject, and    -   b) comparing the amount of said cardiac Troponin as determined        in step a) to a reference amount for the cardiac Troponin, and        the amount of said natriuretic peptide to a reference amount for        the natriuretic peptide, whereby atrial fibrillation is        diagnosed.

The term “atrial fibrillation” is well known in the art. Atrialfibrillation is e.g. reviewed by Fuster et al. which herewith isincorporated by reference in its entire disclosure content (Fuster V,Rydén L E, Asinger R W, et al. ACC/AHA/ESC Guidelines for the Managementof Patients With Atrial Fibrillation: Executive Summary A Report of theAmerican College of Cardiology/American Heart Association Task Force onPractice Guidelines and the European Society of Cardiology Committee forPractice Guidelines and Policy Conferences (Committee to DevelopGuidelines for the Management of Patients With Atrial Fibrillation)Developed in Collaboration With the North American Society of Pacing andElectrophysiology. Circulation. Oct. 23 2001; 104(17):2118-50). Atrialfibrillation is an abnormal heart rhythm which involves the two upperchambers of the heart. In a normal heart rhythm, the impulse generatedby the sino-atrial node spreads through the heart and causes contractionof the heart muscle and pumping of blood. In atrial fibrillation, theregular electrical impulses of the sino-atrial node are replaced bydisorganized, rapid electrical impulses which result in irregular heartbeats.

Atrial fibrillation (AF) can be permanent, persistent or intermittent(for an explanation of these terms, see also Fuster et al. (loc. cit.).

A subject, preferably, suffers from permanent AF, if the AF haspersisted for more than one year. In particular, conversion back tosinus rhythm does not occur (or only if treated).

A subject, preferably, suffers from persistent AF, if the AF lasts morethan 7 days and may require either pharmacologic or electricalintervention to terminate atrial fibrillation. Thus persistent AF occursin episodes, but the arrhythmia does not convert back to sinus rhythmspontaneously.

A subject, preferably, suffers from intermittent AF (frequently alsoreferred to as paroxymal AF), if there are episodes of atrialfibrillation that terminate spontaneously. The episodes of atrialfibrillation may last from seconds to days. Preferably, the episodeslast less than one hour.

In the context of the aforementioned method, preferably, intermittentatrial fibrillation is diagnosed.

Permanent and persistent atrial fibrillation can be easily diagnosed,e.g., on an electrocardiogram. Characteristic findings are, preferably,the absence of P waves, unorganized electrical activity in their place,and irregularity of R-R interval due to irregular conduction of impulsesto the ventricles. Intermittent atrial fibrillation is more difficult todiagnose, since a diagnosis is only possible during the episode ofatrial fibrillation.

The inventors have surprisingly found that the determination of acardiac Troponin (and, optionally of natriuretic peptide) in a sample ofa subject suspected to suffer from atrial fibrillation allows for thediagnosis of atrial fibrillation. In particular, increased levels of acardiac Troponin are indicative for a subject suffering AF, whereasdecreased levels of a cardiac Troponin are indicative for a subject notsuffering from AF. Further, the determination of a cardiac Troponin alsoallows for the diagnosis of intermittent AF, even in the absence of anepisode of AF (at the time point at which the sample is obtained). Thus,by carrying out the aforementioned method, intermittent atrialfibrillation is, preferably, diagnosed in the absence of an episode ofatrial fibrillation, in particular at the time point at which the sampleis obtained. Thus, the subject, preferably, does not suffer from anepisode of AF when the sample is obtained.

The subject in accordance with the aforementioned method of the presentdisclosure shall be suspected to suffer from atrial fibrillation. Asubject suspected to suffer from atrial fibrillation (e.g. fromintermittent atrial fibrillation), preferably, is a subject who has oneor more risk factors of atrial fibrillation. These risk factors are wellknown in the art and include heart disease, including valve problems anda history of heart attack and heart surgery, systemic hypertension,especially if it's not well controlled with lifestyle changes ormedications, and alcohol consumption. Preferably, the subject beingsuspected to belongs to a risk group. In particular, it is envisagedthat the subject is a subject with proven or suspected cardiac disordersincluding subjects having risk factors predisposing to cardiac disorderssuch as arterial or systemic hypertension, diabetes mellitus, smokers,individuals with hyperlipemia or signs of the metabolic syndrome, inparticular if the subject is at advanced age (more than 60, 65, 70 andpreferably 75 years of age). Alternatively, or additionally the subjectmay, preferably, suffer from valvular disorders, preferably from mitralvalve disorders. It is further envisaged that the subject suspected tosuffer from AF, suffers from hyperthyroidism.

In some embodiments of the method of the present disclosure, the subjectwho is suspected to suffer from atrial fibrillation, preferably, suffersfrom ischemic stroke, in particular from cardioembolic ischemic stroke(for an explanation of the these terms, see elsewhere herein). If thesubject to be tested in accordance with the aforementioned methodsuffers from ischemic stroke, the sample is, preferably, obtainedimmediately after the onset of stroke symptoms as described in thecontext of the method for early differentiating whether a subjectsuffers from cardioembolic stroke or from non-cardioembolic ischemicstroke.

However, it is also preferred that the subject to be tested does notsuffer from ischemic stroke.

Preferably, the reference amount in connection with the aforementionedmethod is derived from a subject known to suffer from atrialfibrillation (or from a group of subjects), and wherein an identicalamount of the cardiac Troponin (and, optionally, of the natriureticpeptide), or an amount of the cardiac Troponin (and, optionally, of thenatriuretic peptide) which is increased as compared to the referenceamount, indicates that the subject suffers from atrial fibrillation.Additionally or alternatively, the reference amount is derived from asubject known not to suffer from atrial fibrillation or from a group ofsuch subjects, wherein an identical amount of the cardiac Troponin (and,optionally, of the natriuretic peptide), or an amount of the cardiactroponin Troponin (and, optionally, of the natriuretic peptide) which isdecreased as compared to the reference amount, indicates that thesubject does not suffer from atrial fibrillation.

Further, the reference amount may define a threshold amount for thecardiac Troponin (and, optionally for the natriuretic peptide), wherebyan amount of Troponin (and, optionally of the natriuretic peptide) inthe sample of the test subject larger than the respective thresholdshall be indicative for atrial fibrillation, while an amount of Troponin(and, optionally, of the natriuretic peptide) in the sample of the testsubject lower than the respective threshold shall indicate that thesubject does not suffer from atrial fibrillation.

Exemplary reference amounts are indicated herein below.

A preferred reference amount indicating atrial fibrillation is an amountof a cardiac troponin, in particular of Troponin T of about 6 pg/ml toabout 40 pg/ml and, more preferably, 8 to 30 pg/ml, or about 10 to about20 pg/ml, even more preferably of about 15 to 20 pg/ml. Even morepreferably, the reference is an amount of about 10 pg/ml, or mostpreferably, about 7 pg/ml. A test amount being essentially identical orincreased shall be indicative for atrial fibrillation while a decreasedamount in the test sample as compared to the reference amount shallindicate that the subject does not suffer from atrial fibrillation.Preferably, the aforementioned reference amounts are derived from asubject or a group of subjects known to suffer from atrial fibrillation.

An exemplary reference amount indicating atrial fibrillation is anamount of a natriuretic peptide, in particular, of NT-proBNP of about300 pg/ml to about 1500 pg/ml and, more preferably, 400 to 1300 pg/ml,even more preferably of about 500 to 1000 pg/ml. Even more preferably,the reference is an amount of about 500, 400, or most preferably, ofabout 300 pg/ml. A test amount being essentially identical or increasedshall be indicative for atrial fibrillation while a decreased amount inthe test sample as compared to the reference amount shall indicate thatthe subject does not suffer from atrial fibrillation. Preferably, theaforementioned reference amounts are derived from a subject or a groupof subjects known to suffer from atrial fibrillation.

As set forth above, the diagnosis of intermittent atrial fibrillation ischallenging, whereas the diagnosis of permanent or persistent atrialfibrillation is rather easy. Since subjects with permanent andpersistent atrial fibrillation can be identified without further ado, itis of particular interest to identify those subjects who do not sufferfrom permanent or persistent atrial fibrillation, but who suffer fromintermittent atrial fibrillation.

Interestingly, it has been shown in the context of the studies of thepresent disclosure, that the levels of cardiac troponins and ofnatriuretic peptides are lower in subjects with intermittent atrialfibrillation than in subjects with persistent or permanent atrialfibrillation. This is advantageous, since the determination of a cardiacTroponin and, optionally, a natriuretic peptide allows to identify thosepatients who suffer from intermittent fibrillation.

Therefore, in a preferred embodiment of the present disclosureintermittent atrial fibrillation shall be diagnosed, in particular insubject suspected to suffer from intermittent atrial fibrillation.

In order to allow for the diagnosis of intermittent AF, the amount ofthe cardiac Troponin, and, optionally, the amount of the natriureticpeptide, as determined in step a) shall be compared in step b) to tworeference amounts. It is envisaged that a first reference amount isderived from a subject known to suffer from intermittent AF (or from agroup of such subjects), and that a second reference amount is derivedfrom a subject known to suffer from permanent or, in particular, frompersistent AF (or from a group of such subjects). Of course, thereference amounts shall be derived from samples of the aforementionedsubjects.

Preferably, an amount of the cardiac Troponin and, optionally, of thenatriuretic peptide in the sample of the test subject which isessentially identical or larger than the than the reference amount (forthe cardiac Troponin and, optionally, for the natriuretic peptide)derived from a subject known to suffer from intermittent AF (or from agroup of such subjects), but which is lower than the reference amountderived from a subject known to suffer from permanent or from persistentAF (or from a group of such subjects) is indicative for the diagnosis ofintermittent AF.

A preferred reference amount for a cardiac Troponin, in particular forTroponin T, derived from a subject known to suffer from intermittent AF(or from a group of such subjects) is within a range of about 5 to 10pg/ml. Preferably, the reference amount is about 9 pg/ml.

A preferred reference amount for a cardiac Troponin, in particular forTroponin T, derived from a subject known to suffer from permanent orfrom persistent AF (or from a group of such subjects) is within a rangeof about 12 to 25 pg/ml. Preferably, the reference amount is about 18pg/ml.

A preferred reference amount for a natriuretic peptide, in particularfor NT-proBNP, derived from a subject known to suffer from intermittentAF (or from a group of such subjects) is within a range of about 300 to500 pg/ml. Preferably, the reference amount is about 350 pg/ml.

A preferred reference amount for a natriuretic peptide, in particularfor NT-proBNP, derived from a subject known to suffer from permanent orfrom persistent AF (or from a group of such subjects) is within a rangeof about 900 to 1500 pg/ml. Preferably, the reference amount is about900 pg/ml.

In another preferred embodiment of the method of diagnosing AF,intermittent AF is diagnosed. The subject according to this preferredembodiment shall be known not to suffer from permanent and/or persistentatrial fibrillation (which can be determined without further ado, seeabove).

Thus also envisaged is method for diagnosing intermittent atrialfibrillation in a subject suspected to suffer from intermittent atrialfibrillation, but known not to suffer from persistent and/or permanentatrial fibrillation, comprising

-   -   a) determining the amount of a cardiac Troponin (and,        optionally, a natriuretic peptide) in a sample from said        subject, and    -   b) comparing the, thus, determined amount of said cardiac        Troponin (and, optionally of said natriuretic peptide) to a        reference amount (s), whereby intermittent atrial fibrillation        is diagnosed.

The subject suspected to suffer from intermittent AF, preferably, hasthe same risk factors as the subject suspected to suffer from AF (seeelsewhere herein). In particular, it is envisaged that the subjectsuffers from ischemic stroke, in particular from cardioembolic ischemicstroke.

The reference amount to be applied in the context of the presentdisclosure shall be derived from a subject known to suffer fromintermittent AF, or from a subject known not to suffer from AF.

Preferably, the reference amount in connection with the aforementionedembodiment is derived from a subject known to suffer from intermittentatrial fibrillation (or from a group of subjects), and wherein anidentical amount of the cardiac Troponin (and, optionally, of thenatriuretic peptide), or an amount of the cardiac Troponin (and,optionally, of the natriuretic peptide) which is increased as comparedto the reference amount, indicates that the subject suffers intermittentatrial fibrillation. Additionally or alternatively, the reference amountis derived from a subject known not to suffer from atrial fibrillation,wherein an identical amount of the cardiac Troponin (and, optionally, ofthe natriuretic peptide), or an amount of the cardiac troponin Troponin(and, optionally, of the natriuretic peptide) which is decreased ascompared to the reference amount, indicates that subject does not sufferfrom intermittent atrial fibrillation.

A preferred reference amount indicating intermittent atrial fibrillationis an amount of a cardiac troponin, in particular of Troponin T of about5 pg/ml to about 30 pg/ml and, more preferably, 5 to 25 pg/ml, or about6 to about 10 pg/ml, even more preferably of about 6 to 8 pg/ml. Evenmore preferably, the reference is an amount of about 8 pg/ml, or mostpreferably, about 7 pg/ml. A test amount being essentially identical orincreased shall be indicative for intermittent atrial fibrillation whilea decreased amount in the test sample as compared to the referenceamount shall indicate that the subject does not suffer from intermittentatrial fibrillation. Preferably, the aforementioned reference amountsare derived from a subject or a group of subjects known to suffer fromintermittent atrial fibrillation.

A preferred reference amount indicating intermittent atrial fibrillationis an amount of a natriuretic peptide, in particular, of NT-proBNP ofabout 300 pg/ml to about 800 pg/ml and, more preferably, 300 to 700pg/ml, even more preferably of about 350 to 500 pg/ml. Even morepreferably, the reference is an amount of about 500, 400, or mostpreferably, of about 300 pg/ml. A test amount being essentiallyidentical or increased shall be indicative for intermittent atrialfibrillation while a decreased amount in the test sample as compared tothe reference amount shall indicate that the subject does not sufferfrom intermittent atrial fibrillation. Preferably, the aforementionedreference amounts are derived from a subject or a group of subjectsknown to suffer from intermittent atrial fibrillation.

In a preferred embodiment of the aforementioned method of the presentdisclosure, said method further comprises recommending a therapy forsaid subject, if the subject has been diagnosed to suffer atrialfibrillation, in particular from intermittent atrial fibrillation.Preferred therapies that can be recommended in a subject who suffersfrom atrial fibrillation are, e.g., described by Fuster et al. (Fusteret al. J Am Coll Cardiol 2001: 38: 1231, and Fuster V. et al.Circulation 2006: 114 to 257). Preferred therapies included,administration of beta blockers, non-dihydropyridine calcium channelblockers, digoxin, vitamin K antatgonists, aspirin, acetylsalicylicacid. Moreover, pharmacologic or electrical intervention can berecommended to terminate atrial fibrillation. Pharmacologicintervention, preferably, include administration of flecainide,dofetilide, propafenone and/or ibutilide. Further envisaged is theadministration of factor Xa inhibitors such as rivaroxoban and/ordabigatran (see Patel M. R. et al, NEJM 2011: 365: 883-91; Connolly S.J. et al NEJM 2010: 261: 1139-51).

Preferred embodiments of the aforementioned method.

Preferably, intermittent atrial fibrillation is diagnosed.

In a preferred embodiment, the subject does not suffer from ischemicstroke.

In another preferred embodiment, the subject suspected to suffer fromatrial fibrillation suffers from ischemic stroke, in particular fromcardioembolic stroke, and wherein the sample has been obtainedimmediately after the onset of symptoms of ischemic stroke. Preferably,the sample from said subject has been obtained from said subject notmore than 12 hours after the onset of symptoms of ischemic stroke, inparticular not more than 6 hours or not more than 3 hours after theonset of symptoms of ischemic stroke.

In a preferred embodiment, wherein the subject is known not to sufferfrom persistent and/or permanent atrial fibrillation, in particular,wherein the reference amount is derived from a subject known to sufferfrom intermittent atrial fibrillation or from a group of such subjects,and wherein an identical amount of the cardiac Troponin, or an amount ofthe cardiac Troponin which is increased as compared to the referenceamount, indicates that the subject suffers intermittent atrialfibrillation, and/or wherein the reference amount is derived from asubject known not to suffer from atrial fibrillation or from a group ofsuch subjects, wherein an identical amount of the cardiac Troponin, oran amount of the cardiac troponin Troponin which is decreased ascompared to the reference amount, indicates that subject does not sufferfrom intermittent atrial fibrillation.

In another preferred embodiment, the amount of the cardiac Troponin inthe sample from the subject is compared to two reference amounts,wherein the first reference amount is derived from a subject known tosuffer from intermittent AF or from a group of such subjects, andwherein the second reference amount is derived from a subject known tosuffer from permanent or from persistent AF or from a group of suchsubjects. Preferably, an amount of the cardiac Troponin in the sample ofthe subject which is essentially identical or larger than the than thefirst reference amount, but which is lower than the second referenceamount derived from a subject known to suffer from permanent or frompersistent AF is indicative for the diagnosis of intermittent AF.

In a preferred embodiment, the method further comprises thedetermination of the amount of a natriuretic peptide, in particular of abrain natriuretic peptide, in particular of BNP and NT-proBNP.

Preferably, intermittent atrial fibrillation is diagnosed in the absenceof an episode of atrial fibrillation, in particular at the time point atwhich the sample is obtained.

In another aspect of the disclosure, a system for diagnosing AF, inparticular, intermittent AF, is contemplated, comprising:

a) an analyzer unit configured to bringing a sample from a subject whois suspected to suffer from AF, in particular, intermittent AF intocontact with a detection agent that specifically binds to the markercardiac Troponin for a time sufficient to allow for the formation of acomplex of the said detection agent and the marker from the sample,

-   -   b) an analyzer unit configured to measure the amount of the        formed complex, wherein the said amount of the formed complex is        proportional to the amount of the marker present in the sample,    -   c) a computing device having a processor and in operable        communication with said analysis units, and    -   d) a non-transient machine readable media including a plurality        of instructions executable by the processor, the instructions,        when executed transform the amount of the formed complex into an        amount of the marker reflecting the amount of the marker present        in the sample, compare said amount to a reference, and establish        an aid for diagnosing AF, in particular, intermittent AF based        on the result of said comparison to said reference.

Preferably, the system also comprises a detection agent thatspecifically binds to natriuretic peptide.

In yet an aspect of the aforementioned method, step a) may be carriedout by an analyzer unit, in an aspect, an analyzer unit as definedelsewhere herein.

In an aspect of the method of the disclosure, the amount determined instep a) is compared to a reference. In an aspect, the reference is areference as defined elsewhere herein. In yet another aspect, thereference takes into account the proportional relationship between themeasured amount of complex and the amount present in the originalsample. Thus, the references applied in an aspect of the method of thedisclosure are artificial references which are adopted to reflect thelimitations of the detection agent that has been used. In anotheraspect, said relationship can be also taken into account when carryingout the comparison, e.g., by including a normalization and/or correctioncalculation step for the determined amount prior to actually comparingthe value of the determined amount and the reference. Again, thenormalization and/or correction calculation step for the determinedamount adopts the comparison step such that the limitations of thedetection agent that has been used are reflected properly. In an aspect,the comparison is carried out automatically, e.g., assisted by acomputer system or the like.

The aid for diagnosing AF, in particular, intermittent AF is establishedbased on the comparison carried out in step b) by allocating the subjecteither (i) the group of subjects who suffer from AF or (ii) the group ofwho do not suffer from AF as set forth herein elsewhere. As discussedelsewhere herein already, the allocation of the investigated subjectmust not be correct in 100% of the investigated cases. Moreover, thegroups of subjects into which the investigated subject is allocated areartificial groups in that they are established based on statisticalconsiderations, i.e. a certain preselected degree of likelihood based onwhich the method of the disclosure shall operate. In an aspect of thedisclosure, the aid for optimizing a risk assessment is establishedautomatically, e.g., assisted by a computing device or the like, asdescribed and disclosed herein.

In an aspect of the method of the disclosure, said method furthercomprises a step of recommending and/or managing the subject accordingto the result established in step c) as set forth elsewhere herein indetail.

In an aspect of the aforementioned method, steps b) and/or c) arecarried out by one or more analyzer units as set forth elsewhere herein.

Moreover, the present disclosure relates to the use of a cardiacTroponin and/or of a detection agent, which specifically binds thereto(and optionally of a natriuretic peptide and/or of detection agent,which specifically binds thereto) in a sample of a subject sufferingfrom ischemic stroke for early differentiating whether the subjectsuffers from cardioembolic ischemic stroke or from non-cardioembolicischemic stroke, wherein the sample has been obtained immediately afterthe onset of symptoms of ischemic stroke. Preferably, said sample hasbeen obtained not more than 6 hours after the onset of symptoms ofstroke.

Also envisaged by the present disclosure is the use of a cardiacTroponin and/or of a detection agent, which specifically binds thereto(and optionally of a natriuretic peptide and/or of detection agent,which specifically binds thereto) in a sample of a subject suspected tosuffer from atrial fibrillation for diagnosing atrial fibrillation in asaid subject. Preferably, intermittent atrial fibrillation is diagnosed.

The term “detection agent” as used herein refers to an agent which iscapable of specifically recognizing and binding the biomarker referredto herein (a cardiac Troponin, or a natriuretic peptide) when present ina sample. Moreover, said agent shall allow for direct or indirectdetection of the complex formed by the said agent and the biomarker.Direct detection can be achieved by including into the agent adetectable label. Indirect labelling may be achieved by a further agentwhich specifically binds to the complex comprising the biomarker and thedetection agent wherein the said further agent is than capable ofgenerating a detectable signal. Suitable compounds which can be used asdetection agents are well known in the art. Preferably, the detectionagent is an antibody or aptamere which specifically binds to thebiomarker. Antibodies as referred to herein include both polyclonal andmonoclonal antibodies, as well as fragments thereof, such as Fv, Fab andF(ab)₂ fragments that are capable of binding antigen or hapten. Alsoenvisaged are single chain antibodies and humanized hybrid antibodieswherein amino acid sequences of a non-human donor antibody exhibiting adesired antigen-specificity are combined with sequences of a humanacceptor antibody.

The present disclosure further relates to a device for earlydifferentiating whether a subject suffers from cardioembolic ischemicstroke or from non-cardioembolic ischemic stroke in a subject sufferingfrom ischemic stroke, said device comprising:

a) an analyzing unit (or an analyzer unit) comprising a detection agentfor a cardiac Troponin which allows for the determination of the amountof said cardiac Troponin (and, optionally, a detection agent for anatriuretic peptide which allows for the determination of the amount ofsaid natriuretic peptide); and

-   -   b) an evaluation unit (or an analyzer unit) comprising a data        processor having implemented an algorithm for comparing the        amount(s) determined by the analyzing unit with reference        amount(s) stored in a database in order to differentiate whether        a subject suffers from cardioembolic ischemic stroke or from        non-cardioembolic ischemic stroke, wherein the reference amount        is derived from a sample from a reference subject as described        herein elsewhere in the context of the method for        differentiating whether a subject suffers from cardioembolic        ischemic stroke or from non-cardioembolic ischemic stroke, and        the algorithm is an algorithm as defined in the context of the        said method.

The present disclosure further relates to a device for diagnosing atrialfibrillation, in particular, intermittent atrial fibrillation in asubject suspected to suffer from atrial fibrillation, said devicecomprising:

a) an analyzing unit (or an analyzer unit) comprising a detection agentfor a cardiac Troponin which allows for the determination of the amountof said cardiac Troponin (and, optionally, a detection agent for anatriuretic peptide which allows for the determination of the amount ofsaid natriuretic peptide); and

-   -   b) an evaluation unit (or an analyzer unit) comprising a data        processor having implemented an algorithm for comparing the        amount(s) determined by the analyzing unit with reference        amount(s) stored in a database in order to diagnose atrial        fibrillation, in particular intermittent atrial fibrillation,        wherein the reference amount is derived from a sample from a        reference subject as described herein elsewhere in the context        of the method for diagnosing atrial fibrillation (in particular,        intermittent atrial fibrillation), and the algorithm is an        algorithm as defined in the context of the method for diagnosing        atrial fibrillation (in particular atrial fibrillation).

The term “device” as used herein relates to a system comprising theaforementioned units operatively linked to each other as to allow thediagnosis according to the methods of the disclosure. Preferreddetection agents which can be used for the analyzing unit are disclosedelsewhere herein. The analyzing unit, preferably, comprises saiddetection agents in immobilized form on a solid support which is to becontacted to the sample comprising the biomarkers the amount of which isto be determined. Moreover, the analyzing unit can also comprise adetector which determines the amount of detection agent which isspecifically bound to the biomarker(s). The determined amount can betransmitted to the evaluation unit. Said evaluation unit comprises adata processing element, such as a computer, with an implementedalgorithm for carrying out a comparison between the determined amountand a suitable reference. Suitable references can be derived fromsamples of subjects to be used for the generation of reference amountsas described elsewhere herein above. The diagnostic results may be givenas output of parametric diagnostic raw data, preferably, as absolute orrelative amounts. It is to be understood that these data may needinterpretation by the clinician. However, also envisage are expertsystem devices wherein the output comprises processed diagnostic rawdata the interpretation of which does not require a specializedclinician. Preferably, the device of the present disclosure can be usedto carry out the aforementioned method of the present disclosure in anautomated manner.

A preferred embodiment of the instant disclosure includes a system forguiding exercise as set forth herein. Examples of systems includeclinical chemistry analyzers, coagulation chemistry analyzers,immunochemistry analyzers, urine analyzers, nucleic acid analyzers, usedto detect the result of chemical or biological reactions or to monitorthe progress of chemical or biological reactions. More specifically,exemplary systems of the instant disclosure may include Roche Elecsys™Systems and Cobas® e Immunoassay Analyzers, Abbott Architect™ and Axsym™Analyzers, Siemens Centaur™ and Immulite™ Analyzers, and Beckman CoulterUniCel™ and Acess™ Analyzers, or the like.

Embodiments of the system may include one or more analyzer unitsutilized for practicing the subject disclosure. The analyzer units ofthe system disclosed herein are in operable communication with thecomputing device disclosed herein through any of a wired connection,Bluetooth, LANS, or wireless signal, as are known. Additionally,according to the instant disclosure, an analyzer unit may comprise astand-alone apparatus, or module within a larger instrument, whichperforms one or both of the detection, e.g. qualitative and/orquantitative evaluation of samples for diagnostic purpose. For example,an analyzer unit may perform or assist with the pipetting, dosing,mixing of samples and/or reagents. An analyzer unit may comprise areagent holding unit for holding reagents to perform the assays.Reagents may be arranged for example in the form of containers orcassettes containing individual reagents or group of reagents, placed inappropriate receptacles or positions within a storage compartment orconveyor. Detection reagents may also be in immobilized form on a solidsupport which are contacted with the sample. Further, an analyzer unitmay include a process and/or detection component which is optimizablefor specific analysis.

According to some embodiments, an analyzer unit may be configured foroptical detection of an analyte, for example a marker, with a sample. Anexemplary analyzer unit configured for optical detection comprises adevice configured for converting electro-magnetic energy into anelectrical signal, which includes both single and multi-element or arrayoptical detectors. According to the present disclosure, an opticaldetector is capable of monitoring an optical electro-magnetic signal andproviding an electrical outlet signal or response signal relative to abaseline signal indicative of the presence and/or concentration of ananalyte in a sample being located in an optical path. Such devices mayalso include, for example, photodiodes, including avalanche photodiodes,phototransistors, photoconductive detectors, linear sensor arrays, CCDdetectors, CMOS detectors, including CMOS array detectors,photomultipliers, and photomultiplier arrays. According to certainembodiments, an optical detector, such as a photodiode orphotomultiplier, may contain additional signal conditioning orprocessing electronics. For example, an optical detector may include atleast one pre-amplifier, electronic filter, or integrated circuit.Suitable pre-preamplifiers include, for example, integrating,transimpedance, and current gain (current mirror) pre-amplifiers.

Additionally, one or more analyzer unit according to the instantdisclosure may comprise a light source for emitting light. For example,a light source of an analyzer unit may consist of at least one lightemitting element (such as a light emitting diode, an electric poweredradiation source such as an incandescent lamp, an electroluminescentlamp, a gas discharge lamp, a high-intensity discharge lamp, a laser)for measuring analyte concentrations with a sample being tested or forenabling an energy transfer (for example, through florescent resonanceenergy transfer or catalyzing an enzyme).

Further, an analyzer unit of the system may include one or moreincubation units (for example, for maintaining a sample or a reagent ata specified temperature or temperature range). In some embodiments, ananalyzer unit may include a thermocycler, include a real-timethermocycler, for subjecting a sample to repeated temperature cycles andmonitoring a change in the amount of an amplification product with thesample.

Additionally, an analyzer unit of the system disclosed herein maycomprise, or be operationally connected to, a reaction vessel or cuvettefeeding unit. Exemplary feeding units include liquid processing units,such as a pipetting unit, to deliver samples and/or reagents to thereaction vessels. The pipetting unit may comprise a reusable washableneedle, e.g. a steel needle, or disposable pipette tips. The analyzerunit may further comprise one or more mixing units, for example a shakerto shake a cuvette comprising a liquid, or a mixing paddle to mixliquids in a cuvette, or reagent container.

It follows from the above that according to some embodiments of theinstant disclosure, portions of some steps of methods disclosed anddescribed herein may be performed by a computing device. A computingdevice may be a general purpose computer or a portable computing device,for example. It should also be understood that multiple computingdevices may be used together, such as over a network or other methods oftransferring data, for performing one or more steps of the methodsdisclosed herein. Exemplary computing devices include desktop computers,laptop computers, personal data assistants (“FDA”), such as BLACKBERRYbrand devices, cellular devices, tablet computers, servers, and thelike. In general, a computing device comprises a processor capable ofexecuting a plurality of instructions (such as a program of software).

A computing device has access to a memory. A memory is a computerreadable medium and may comprise a single storage device or multiplestorage devices, located either locally with the computing device oraccessible to the computing device across a network, for example.Computer-readable media may be any available media that can be accessedby the computing device and includes both volatile and non-volatilemedia. Further, computer readable-media may be one or both of removableand non-removable media. By way of example, and not limitation,computer-readable media may comprise computer storage media. Exemplarycomputer storage media includes, but is not limited to, RAM, ROM,EEPROM, flash memory or any other memory technology, CD-ROM, DigitalVersatile Disk (DVD) or other optical disk storage, magnetic cassettes,magnetic tape, magnetic disk storage or other magnetic storage devices,or any other medium which can be used for storing a plurality ofinstructions capable of being accessed by the computing device andexecuted by the processor of the computing device.

According to embodiments of the instant disclosure, software may includeinstructions which, when executed by a processor of the computingdevice, may perform one or more steps of the methods disclosed herein.Some of the instructions may be adapted to produce signals that controloperation of other machines and thus may operate through those controlsignals to transform materials far removed from the computer itself.These descriptions and representations are the means used by thoseskilled in the art of data processing, for example, to most effectivelyconvey the substance of their work to others skilled in the art.

The plurality of instructions may also comprise an algorithm which isgenerally conceived to be a self-consistent sequence of steps leading toa desired result. These steps are those requiring physical manipulationsof physical quantities. Usually, though not necessarily, thesequantities take the form of electrical or magnetic pulses or signalscapable of being stored, transferred, transformed, combined, compared,and otherwise manipulated. It proves convenient at times, principallyfor reasons of common usage, to refer to these signals as values,characters, display data, numbers, or the like as a reference to thephysical items or manifestations in which such signals are embodied orexpressed. It should be borne in mind, however, that all of these andsimilar terms are to be associated with the appropriate physicalquantities and are merely used here as convenient labels applied tothese quantities. According to some embodiments of the instantdisclosure, an algorithm for carrying out a comparison between adetermined amount of one or more markers disclosed herein, and asuitable reference, is embodied and performed by executing theinstructions. The results may be given as output of parametricdiagnostic raw data or as absolute or relative amounts. According tovarious embodiments of the system disclosed herein, a “diagnosis” may beprovided by the computing device of a system disclosed herein based onsaid comparison of the calculated “amount” to a reference or athreshold. For example, a computing device of a system may provide anindicator, in the form of a word, symbol, or numerical value which isindicative of a particular diagnosis.

The computing device may also have access to an output device. Exemplaryoutput devices include fax machines, displays, printers, and files, forexample. According to some embodiments of the present disclosure, acomputing device may perform one or more steps of a method disclosedherein, and thereafter provide an output, via an output device, relatingto a result, indication, ratio or other factor of the method.

The present disclosure further encompasses a kit for earlydifferentiating whether a subject suffers from cardioembolic ischemicstroke or from non-cardioembolic ischemic stroke in a subject suffersfrom ischemic stroke, said kit comprising a detection agent for acardiac Troponin and a standard (standards) which reflects the referenceamount(s) as derived from a sample from a subject known suffer fromcardioembolic stroke and/or from a subject known to suffer fromnon-cardioembolic stroke.

The present disclosure, finally, encompasses a kit for diagnosing atrialfibrillation in a subject suspected to suffer from atrial fibrillation,said kit comprising a detection agent for a cardiac Troponin (and,optionally, for a natriuretic peptide) and a standard (standards) whichreflects the reference amount(s) as derived from a sample from a subjectknown suffer from atrial fibrillation and/or from a subject known not tosuffer from atrial fibrillation.

The term “kit” as used herein refers to a collection of theaforementioned components, preferably, provided in separately or withina single container. The container also comprises instructions forcarrying out the method of the present disclosure. These instructionsmay be in the form of a manual or may be provided by a computer programcode which is capable of carrying out the comparisons referred to in themethods of the present disclosure and to establish a diagnosisaccordingly when implemented on a computer or a data processing device.The computer program code may be provided on a data storage medium ordevice such as a optical storage medium (e.g., a Compact Disc) ordirectly on a computer or data processing device. Moreover, the kit may,preferably, comprise standards which reflect the reference amounts asdescribed and referred to elsewhere herein in detail. The detectionagent is, preferably, immobilized on a carrier, and, preferably, a teststripe

The following examples, sequence listing, and figures are provided forthe purpose of demonstrating various embodiments of the instantdisclosure and aiding in an understanding of the present disclosure, thetrue scope of which is set forth in the appended claims. These examplesare not intended to, and should not be understood as, limiting the scopeor spirit of the instant disclosure in any way. It should also beunderstood that modifications can be made in the procedures set forthwithout departing from the spirit of the disclosure.

ILLUSTRATIVE EMBODIMENTS

The following comprises a list of illustrative embodiments according tothe instant disclosure which represent various embodiments of theinstant disclosure. These illustrative embodiments are not intended tobe exhaustive or limit the disclosure to the precise forms disclosed,but rather, these illustrative embodiments are provided to aide infurther describing the instant disclosure so that others skilled in theart may utilize their teachings.

-   1. A method for early differentiating whether a subject suffers from    cardioembolic ischemic stroke or from non-cardioembolic ischemic    stroke, comprising determining the amount of a cardiac Troponin in a    sample from a subject suffering from ischemic stroke, wherein the    sample has been obtained not more than 6 hours after the onset of    symptoms of ischemic stroke.-   2. The method of embodiment 1, further comprising the step of    comparing the amount of said cardiac Troponin to a reference amount,    thereby differentiating whether said subject suffers from    cardioembolic stroke or from non-cardioembolic ischemic stroke.-   3. The method of embodiments 1 and 2, wherein the sample been    obtained from said subject not more than 3 hours after the onset of    symptoms of ischemic stroke.-   4. The method of any one of embodiments 1 to 3, wherein the    reference amount is derived from a subject known to suffer from    cardioembolic stroke, and wherein an identical amount of the cardiac    Troponin, or an amount of the cardiac troponin which is increased as    compared to the reference amount, indicates that the subject suffers    from cardioembolic stroke, and/or    -   wherein the reference amount is derived from a subject known to        suffer from non-cardioembolic ischemic stroke, and wherein an        identical amount of the cardiac Troponin, or an amount of the        cardiac troponin which is decreased as compared to the reference        amount, indicates that the subject suffers from        non-cardioembolic ischemic stroke.-   5. The method of any one of embodiments 1 to 5, further comprising    the determination of the amount of a natriuretic peptide, in    particular of a brain natriuretic peptide, in particular of BNP or    NT-proBNP.-   6. Use of a cardiac Troponin and/or of a detection agent, which    specifically binds thereto in a sample of a subject for early    differentiating whether the subject suffers from cardioembolic    ischemic stroke or from non-cardioembolic ischemic stroke, wherein    the sample has been obtained not later than 6 hours after the onset    of symptoms of ischemic stroke.-   7. A device for early differentiating whether a subject suffers from    cardioembolic ischemic stroke or from non-cardioembolic ischemic    stroke in a subject suffering from ischemic stroke, said device    comprising:    -   a) an analyzing unit comprising a detection agent for a cardiac        Troponin which allows for the determination of the amount of        said cardiac Troponin (and, optionally, a detection agent for a        natriuretic peptide which allows for the determination of the        amount of said natriuretic peptide); and    -   b) an evaluation unit comprising a data processor having        implemented an algorithm for comparing the amount(s) determined        by the analyzing unit with reference amount(s) stored in a        database in order to differentiate whether a subject suffers        from cardioembolic ischemic stroke or from non-cardioembolic        ischemic stroke, wherein the reference amount is derived from a        sample from a subject known to suffer from cardioembolic stroke        and/or from a sample from a subject known to suffer from        non-cardioembolic ischemic stroke, wherein said sample has been        obtained not more than 6 hours after the onset of symptoms of        ischemic stroke, and wherein the algorithm is as follows:        -   i) an identical amount of the cardiac Troponin, or an amount            of the cardiac troponin which is increased as compared to            the reference amount, is indicative for a subject suffers            from cardioembolic stroke, if the reference amount is            derived from a subject known to suffer from cardioembolic            stroke, and/or        -   ii) an identical amount of the cardiac Troponin, or an            amount of the cardiac troponin which is decreased as            compared to the reference amount, is indicative for a            subject who suffers from non-cardioembolic ischemic stroke,            if the reference amount is derived from a subject known to            suffer from non-cardioembolic ischemic stroke.-   8. A method for diagnosing intermittent atrial fibrillation in a    human subject suspected to suffer from atrial fibrillation,    comprising determining the amount of a cardiac Troponin in a sample    from said subject.-   9. The method of embodiment 8, further comprising the step of    comparing the amount of the cardiac Troponin to a reference amount.-   10. The method of embodiments 8 and 9, wherein the subject does not    suffer from ischemic stroke.-   11. The method of embodiments 8 and 9, wherein the subject suspected    to suffer from atrial fibrillation suffers from ischemic stroke, in    particular from cardioembolic stroke, and wherein the sample has    been obtained immediately after the onset of symptoms of ischemic    stroke.-   12. The method of embodiment 11, wherein said sample from said    subject has been obtained from said subject not more than 12 hours    after the onset of symptoms of ischemic stroke, in particular not    more than 6 hours or not more than 3 hours after the onset of    symptoms of ischemic stroke.-   13. The method of embodiments 8 to 12, wherein the subject is known    not to suffer from persistent and permanent atrial fibrillation.-   14. The method of embodiment 13, wherein the reference amount is    derived from a subject known to suffer from intermittent atrial    fibrillation or from a group of such subjects, and wherein an    identical amount of the cardiac Troponin, or an amount of the    cardiac Troponin which is increased as compared to the reference    amount, indicates that the subject suffers intermittent atrial    fibrillation, and/or wherein the reference amount is derived from a    subject known not to suffer from atrial fibrillation or from a group    of such subjects, wherein an identical amount of the cardiac    Troponin, or an amount of the cardiac troponin Troponin which is    decreased as compared to the reference amount, indicates that    subject does not suffer from intermittent atrial fibrillation.-   15. The method of embodiments 9 to 12, wherein the amount of the    cardiac Troponin in the sample from the subject is compared to two    reference amounts, wherein the first reference amount is derived    from a subject known to suffer from intermittent AF or from a group    of such subjects, and wherein the second reference amount is derived    from a subject known to suffer from permanent or from persistent AF    or from a group of such subjects.-   16. The method of embodiment 15, wherein an amount of the cardiac    Troponin in the sample of the subject which is essentially identical    or larger than the than the first reference amount, but which is    lower than the second reference amount derived from a subject known    to suffer from permanent or from persistent AF is indicative for the    diagnosis of intermittent AF.-   17. The method of any one of embodiments 8 to 16, further comprising    the determination of the amount of a natriuretic peptide, in    particular of a brain natriuretic peptide, in particular of BNP and    NT-proBNP.-   18. The method of any one of embodiments 8 to 17, wherein    intermittent atrial fibrillation is diagnosed in the absence of an    episode of atrial fibrillation, in particular at the time point at    which the sample is obtained.-   19. Use of a cardiac Troponin and/or of a detection agent, which    specifically binds thereto in a sample of a subject for early    differentiating whether the subject suffers from cardioembolic    ischemic stroke or from non-cardioembolic ischemic stroke, wherein    the sample has been obtained not later than 6 hours after the onset    of symptoms of ischemic stroke.-   20. Use of a cardiac Troponin and/or of a detection agent, which    specifically binds thereto in a sample of a human subject suspected    to suffer from atrial fibrillation for diagnosing intermittent    atrial fibrillation in a said subject.-   21. A device for early differentiating whether a subject suffers    from cardioembolic ischemic stroke or from non-cardioembolic    ischemic stroke in a subject suffering from ischemic stroke, said    device comprising:    -   a) an analyzing unit comprising a detection agent for a cardiac        Troponin which allows for the determination of the amount of        said cardiac Troponin (and, optionally, a detection agent for a        natriuretic peptide which allows for the determination of the        amount of said natriuretic peptide); and    -   b) an evaluation unit comprising a data processor having        implemented an algorithm for comparing the amount(s) determined        by the analyzing unit with reference amount(s) stored in a        database in order to differentiate whether a subject suffers        from cardioembolic ischemic stroke or from non-cardioembolic        ischemic stroke, wherein the reference amount is derived from a        sample from a subject defined in embodiment 4, and the algorithm        is an algorithm as defined in embodiment 4.-   22. A device for diagnosing intermittent atrial fibrillation in a    subject suspected to suffer from atrial fibrillation, said device    comprising:    -   a) an analyzing unit comprising a detection agent for a cardiac        Troponin which allows for the determination of the amount of        said cardiac Troponin; and    -   b) an evaluation unit comprising a data processor having        implemented an algorithm for comparing the amount determined by        the analyzing unit with a reference amount (or reference        amounts) stored in a database in order to diagnose intermittent        atrial fibrillation, wherein the reference amount is derived        from a sample from a reference subject as defined in embodiment        14 or 15, and the algorithm is an algorithm as defined in        embodiment 14 or 16.

EXAMPLES Example 1 Determination of Troponin T, GDF-15 and NT-proBNP

Troponin T was determined using Roche's electrochemiluminescence ELISAsandwich test Elecsys Troponin T hs (high sensitive) STAT (Short TurnAround Time) assay. The test employs two monoclonal antibodiesspecifically directed against human cardiac troponin T. The antibodiesrecognize two epitopes (amino acid position 125-131 and 136-147) locatedin the central part of the cardiac troponin T protein, which consists of288 amino acids (analytical sensitivity below 1.0).

NT-proBNP was determined using Roche's electrochemiluminescence ELISAsandwich test Elecsys proBNP II STAT (Short Turn Around Time) assay. Thetest employs two monoclonal antibodies which recognize epitopes locatedin the N-terminal part (1-76) of proBNP (1-108).

To determine the concentration of GDF-15 in serum and plasma samples, anElecsys prototype test was employed, using a polyclonal, GDF-15 affinitychromatography-purified, goat anti-human GDF-15 IgG antibody from R&DSystems (AF957). In each experiment, a standard curve was generated withrecombinant human GDF-15 from R&D Systems (957-GD/CF). The results withnew batches or recombinant GDF-15 protein were tested in standard plasmasamples and any deviation above 10% was corrected by introducing anadjustment factor for this assay. GDF-15 measurements in serum andplasma samples from the same patient yielded virtually identical resultsafter correction for eventual dilution factors. The detection limit ofthe assay was 200 pg/ml.

Example 2 Patient Cohort

A total of 255 patients with ischemic stroke (mean age 70 years) weretested for NT pro BNP, trponinT and GDF 15. Transitory ischemic attackwas present in 23 patients, minor stroke was diagnosed in 61 patientsand major stroke was found in 108 patients. In addition as describedabove caotoid and transcranial ultrasound as well as electro- andechocardiography were performed and the patients were classifiedaccording to the TOAST criteria. Moreover a 7 day HOLTER ECG wasperformed to identify unnoticed atrial fibrillation on the routineelectrocardiogram.

Example 3 Results

The following results were obtained (indicated are the median values,the 25. percentile and the 75. percentile):

Troponin T NT-pro BNP GDF 15 pg/ml pg/ml pg/ml Large Artery stenosis 4,9262 1146 N=46 0,0-16,3 110-611   801-1404 Cardioembolic stroke 11,6  8681393 N=66 3,8-29   365-1863 1005-2481 Small vessel 6,3 222 1188 N=320,0-9,3  79-412  822-2025 Undetermined 5,3 152 1106 N=97 0,0-10,8 63-371 805-1522

As previously described cardioembolic stroke was associated withincreased NT-pro BNP levels, GDF 15 did not contribute significantly tostroke classification, however sensitive troponin T did and therebyexcluding the difficulties of separating cardiac and brain B typenatriuretic peptides.

These data are further supported by the classification related to thepresence of absence of atrial fibrillation. The results are as follows:

Troponin T N T-pro BNP GDF 15 Pg/ml pg/ml pg/ml Atrial fibrillation 15,81773 2220 N=44 8-35   996-2667 1288-3069 Intermittent AF  9,2 448 1364N=28 4,9-24   321-802  1120-2214 No AF N=101  4,3 137 1069 0-10,6 62-386  765-1480

These data again demonstrate the association of AF with NT-pro BNP andtropinin T but to a much lower extent with GDF 15. The limitations ofNT-pro BNP to be used in the classification were also supported by thefact that median NT-pro BNP levels increased from presentation (331pg/ml) to 24 h follow up to 437 pg/ml, which is in the range of 30%increase. To what extent this increase was due to cardiac causes orreleased from the brain is not clear. In contrast, there was nosignificant increase of Troponin T in the follow-up. Thus, Troponin Tlevels remained stable.

In summary troponin T was found to be a powerful tool in theidentification/separation of causes of stroke. This method can also beused in stroke prevention in combination with B type natriureticpeptides. GDF 15 provided surprisingly little additional information tothis important clinical question.

Example 4 Case Studies

A 68 year old male is diagnosed with TIA (transient ischmemic attack)based on clinical symptoms and a subsequent MRI. His ECG is normal. Histroponin T is 10.2 pg/ml, his NT-pro BNP is 520 pg/ml. An echocardiogramshown mild left ventricular dysfunction, in the atria there was nothrombus formation. Based on the TOAST criteria, the subject diagnosedas cardioembolic stroke after ruling out other possibilities. Because ofthe intraatrial thrombus formation he receives a Holter ECG for 3 dayswhich reveals paroxysmal atrial fibrillation. He is then places onanticoagulant therapy as he had no contraindications.

A 72 year old male presents with minor stroke confirmed by MRI afterruling out intracerebral bleeding by CT scan. His Troponin T is 4.1pg/ml and his NT-pro BNP is 245 pg/ml. A Carotis ultrasound reveals a80% stenosis of the right carotis fork. ECG and echocardiography arenormal, except minor diastolic dysfunction. As he is unlikely to have AFa Holter ECG was not carried out. He is advised to consider revision ofthe obstructed carotis after more intense evaluation of intra- andextracerebral arteries.

A 52 year old female reports dizzyness and palpitations and visits theemergency room. Her Troponin T is 3 pg/ml, NT-pro BNP is 115 pg/ml, theECG and the echocardiography are within normal. Because symptoms do notdirect to a cerebral event or atrial fibrillation no furtherinvestigations were done which is in concordance with the troponin T andNT-pro BNP results. She was discharged with a suspected anxietysyndrome.

A 58 year male presents to the emergency room because of temporaryweakness of his left arm. His ECG is normal, his Troponin T is 11.1pg/ml and NT-pro BNP is 435 pg/ml. A MRI rules out stroke, because ofthe Troponin T and NT-pro BNP results a Holter ECG was later performedwhich revealed paroxysmal AF. A subsequent Echo including TTE revealedintraatrial thrombi. He is diagnosed with paroxysmal atrial fibrillationand put on anticoagulant therapy with no obvious contraindications.

A 58 year old male presents with an ischemic stroke 2 hours after startof symptoms to the emergency room, symptoms include sudden weakness ofthe right arm and leg, his Troponin T is 12.5 pg/ml and NT-pro BNP is920 pg/ml. Suspected cardioembolic stroke is confirmed by esophagealechocardiography with a visible thrombus in the left atrium. Angiographyassociated with lysis therapy confirmed the diagnosis. Lysis therapy wassuccessful and symptoms improved, the patient is placed onanticoagulants.

A 58 year old male presents with dizzyness to his doctor, he haddiabetes mellitus for the past 8 years and he had smoked for the mosttime in this life, arterial hypertension was known for the past 10years. Imaging excludes TIA, on echocardiography he has a dilatated leftatrium without thrombus formation, at presentation his ECG is normal andsinus rhythm was recorded. His troponin is 11 pg/ml, NT-pro BNP is 480pg/ml. A few weeks later, the patient is put on Holter ECG andintermittent atrial fibrillation is recorded.

CONCLUSIONS

The identification of stroke patients with cardioembolic stroke is ofimportance as it directs further diagnostic steps and treatment and evenmore importantly the prevention of future strokes. Natriuretic peptideshave shown usefulness in detecting candidates for cardioembolic strokein patients presenting with ischemic stroke. However, as shown herenatriuretic peptides may change during the course of stroke limitingtheir diagnostic potential, this is not the case with troponin T whichis not subject to substantial change.

A similar reasoning applies to the detection of paroxymal orintermittent atrial fibrillation. Atrial fibrillation is more frequentin the elderly population. Since diagnostic methods (Holter ECG anddetections methods for resulting atrial thrombi, TEE) are of limitedavailability patient selection (rule in/rule out) is important. This canbe achieved by the determination of troponin T (and NT-pro BNP) and theuse of appropriate cut offs.

In conclusion the inventors have identified troponin T as an importantdiagnostic method in ischemic stroke as well as in intermittent atrialfibrillation in order to direct further diagnostic methods and treatmentschedules

All references cited in this specification are herewith incorporated byreference with respect to their entire disclosure content and thedisclosure content specifically mentioned in this specification.

While this disclosure has been described as having an exemplary design,the present disclosure may be further modified within the spirit andscope of this disclosure. This application is therefore intended tocover any variations, uses, or adaptations of the disclosure using itsgeneral principles. Further, this application is intended to cover suchdepartures from the present disclosure as come within the known orcustomary practice in the art to which this disclosure pertains.

What is claimed is:
 1. A method of diagnosing intermittent atrialfibrillation in a patient suffering from atrial fibrillation, the methodcomprising the steps of: contacting, in vitro, a portion of a plasma orserum sample obtained from the patient with a binding agent thatspecifically binds Troponin T, whereby a complex of the binding agentand the Troponin T is formed; separating the complex from binding agentand sample not comprising the complex; measuring the amount of complexseparated in said step of separating, whereby a concentration of theTroponin T in the sample is calculated; comparing the concentration ofthe Troponin T in the sample to a first Troponin reference concentrationand a second Troponin reference concentration, the second Troponinreference concentration being greater than the first Troponin referenceconcentration; and providing a diagnosis of intermittent atrialfibrillation in the patient if the concentration of the Troponin T isgreater than the first Troponin reference concentration and less thanthe Troponin reference concentration.
 2. The method of claim 1, whereinthe binding agent is an antibody.
 3. The method of claim 1, wherein thebinding agent comprises a detectable marker.
 4. The method of claim 3,wherein said step of measuring comprises quantifying a signal from thedetectable label of binding agent comprising the complex.
 5. The methodof claim 1, wherein the sample is obtained from the patient within 6hours of onset of symptoms of atrial fibrillation.
 6. The method ofclaim 1, wherein the second Troponin reference concentration isdetermined from a Troponin reference concentration of a sample obtainedfrom at least one individual previously identified as suffering fromatrial fibrillation.
 7. The method of claim 6, wherein the firstTroponin reference concentration is determined from a Troponin referenceconcentration of a sample obtained from at least one individualpreviously identified as not suffering from atrial fibrillation and notsuffering from intermittent atrial fibrillation.
 8. A method ofdiagnosing intermittent atrial fibrillation in a patient suffering fromatrial fibrillation, the method comprising the steps of: contacting aportion of a serum or plasma sample obtained from the patient with acapture antibody having specific binding affinity for a first epitope ofTroponin T and with a detection antibody having specific bindingaffinity for a second epitope of Troponin T, thereby forming a complexof the capture antibody, Troponin T and the detection antibody, thefirst epitope being different than the second epitope, the detectionantibody having a detectable label; binding the complex of the captureantibody, Troponin T and detection antibody with a solid support;separating the complex of the capture antibody, Troponin T and detectionantibody bound to the solid support from capture antibody, detectionantibody and Troponin T not bound to the solid support; detecting asignal from the detectable label of the detection antibody comprisingthe complex separated from capture antibody, detection antibody andTroponin T not bound to the solid support; quantifying the signaldetected in said step of detecting, the signal being proportional to anamount of Troponin T in the sample from the patient, whereby aconcentration of the Troponin T in the sample is calculated; comparingthe concentration of Troponin T calculated in said step of quantifyingto a first Troponin reference concentration and a second Troponinreference concentration, the second Troponin reference concentrationbeing greater than the first Troponin reference concentration; andproviding a diagnosis of intermittent atrial fibrillation in the patientif the concentration of the Troponin T in the sample calculated in saidstep of quantifying is greater than the first Troponin referenceconcentration and less than the second Troponin reference concentration.9. The method of claim 8, wherein the sample is obtained from thepatient within 6 hours of onset of symptoms of atrial fibrillation. 10.The method of claim 8, wherein the second Troponin referenceconcentration is determined from a Troponin reference concentration of asample obtained from at least one individual previously identified assuffering from atrial fibrillation.
 11. The method of claim 8, whereinthe first Troponin reference concentration is determined from a Troponinreference concentration of a sample obtained from at least oneindividual previously identified as not suffering from atrialfibrillation and not suffering from intermittent atrial fibrillation.